Method and system for tissue treatment with critical/supercritical carbon dioxide

ABSTRACT

Methods of decontaminating bone tissue and an apparatus or system for the same are provided. The methods can be multi-batch processes and include contacting the bone tissue having contaminants with carbon dioxide to decontaminate the bone tissue and to form carbon dioxide having contaminants. The contaminated carbon dioxide is collected and the contaminants are removed to obtain purified carbon dioxide which can be recycled to treat contaminated bone tissue. The contaminated carbon dioxide can be purified by bubbling it through water and/or an organic solvent followed by acid treatment, filtering and liquefying the carbon dioxide. Contaminants that can be removed from contaminated bone tissue, and in turn, from contaminated carbon dioxide include infectious organisms, bacteria, viruses, protozoa, parasites, fungi and mold or a mixture thereof.

BACKGROUND

The rapid and effective repair of bone defects caused by injury,disease, wounds, or surgery is a goal of orthopedic surgery. Toward thisend, a number of compositions and materials have been used or proposedfor use in the repair of bone defects. The biological, physical, andmechanical properties of the compositions and materials are among themajor factors influencing their suitability and performance in variousorthopedic applications.

Autologous cancellous bone (“ACB”), also known as autograft orautogenous bone is considered the gold standard for bone grafts. ACB isosteoinductive and non-immunogenic, and, by definition, has all of theappropriate structural and functional characteristics appropriate forthe particular recipient. Unfortunately, ACB is only available in alimited number of circumstances. Some individuals lack ACB ofappropriate dimensions and quality for transplantation, and donor sitepain and morbidity can pose serious problems for patients and theirphysicians.

Much effort has been invested in the identification or development ofalternative bone graft materials. In the procurement and processing ofxenograft or allograft, a prime consideration is minimizing the risk oftransferring potentially harmful diseases to the bone recipient. Infact, provision of bone tissue safe for transplantation provides a veryspecial challenge as immunogenic material and also microorganisms andviruses can be found deep within the internal matrix of bone samples.

Transplanting of contaminated bone can have serious consequences to therecipient. For example, transmission of human immunodeficiency virus(HIV) via bone grafting is well known. Accordingly, there is a greatneed for bone processing methods that decrease the risk of diseasetransmission associated with the use of, and preparation and procurementof, transplantable bone to the recipient. In this regard it is alsoimportant to recognize that even with a state of the art donor screeningmethodology, recent infections in a particular donor may not bedetected, thereby underscoring the importance of improved cleaning anddecontaminating treatments that offer prophylactic protection againstpotential, or as yet undetected, infectious agents.

Current methods for viral inactivation and sterilization involve the useof toxic chemicals, high temperature and/or irradiation. The harshtreatment of biological active materials such as bone grafting materialscause the degradation or decomposition of materials, destroy biologicalactivity, for example osteoconductivity of demineralized bone tissue,and reduce mechanical properties significantly.

There are also significant limitations on the extent to whichdecontaminating agents have been used successfully to penetrate and todecontaminate the bone matrix. Bone tissue contains potentiallyremovable materials, for example, marrow, cells and lipids that impedeaccess of decontaminating agents deep into bone tissue where infectiousagents or immunogenic macromolecules may be present.

Methods have been developed for treating bone tissue with carbon dioxideas part of critical point dehydration. Other methods have usedsupercritical carbon dioxide to achieve viral inactivation and/orterminal sterilization of bone tissue. Some of these methods requiredlarge amounts of carbon dioxide which have been costly. The carbondioxide used in the treatment of bone tissue would also becomecontaminated with infectious agents and/or other solvents and, whenreleased to the atmosphere, would have a negative environmental impact.Because in some instances the treatment of bone tissue was notautomated, human error would contribute to an inefficient process thatwould provide an inconsistent product.

Accordingly, there is a need for automated, efficient methods oftreating bone tissue without compromising the integrity of desirablebiomaterials present in bone tissue and at the same time reduce costsassociated with the use of large amounts of carbon dioxide, reduce oreven eliminate human error, and provide product consistency whileimproving the environment.

SUMMARY

Methods and systems are provided that allow automation in treating bonewithout comprising the integrity of desirable biomaterials present inbone tissue. Methods of decontaminating bone tissue using carbon dioxideand methods of purifying the contaminated carbon dioxide are provided.This is done to recycle the carbon dioxide so the contaminated carbondioxide can be purified and re-used. The methods described in thisapplication include contacting contaminated bone tissue with carbondioxide to extract the contaminants from the bone tissue and form carbondioxide having contaminants; collecting the carbon dioxide havingcontaminants and separating the contaminants from the collected carbondioxide. In some embodiments, the purified carbon dioxide is recycledfor use in treating the contaminated bone tissue. In other embodiments,the contaminated bone tissue can be treated in multiple batch processeswherein the purified carbon dioxide from one batch can be used to treatcontaminated bone tissue in the next batch.

In some embodiments, the methods of purifying the contaminated carbondioxide obtained from treatment of contaminated bone tissue with carbondioxide include collecting the carbon dioxide having contaminants andseparating the contaminants from the collected contaminated carbondioxide to obtain the purified carbon dioxide. In some embodiments, thepurified carbon dioxide is recycled for use in treating contaminatedbone tissue.

An apparatus or system for treating contaminated bone tissue withpurified carbon dioxide, purifying the contaminated carbon dioxide andrecycling the purified carbon dioxide to treat contaminated bone tissueis also provided. In various embodiments, the system for treatingcontaminated bone tissue with purified carbon dioxide includes a bonetissue chamber configured for holding contaminated bone tissue andreceiving purified carbon dioxide into the bone tissue chamber andevacuating contaminated carbon dioxide from the bone tissue chamber; apurified carbon dioxide supply configured for supplying purified carbondioxide to the bone tissue chamber to decontaminate the bone tissue; acollection chamber configured to receive contaminated carbon dioxidefrom the bone tissue chamber and evacuate contaminated carbon dioxidefrom the collection chamber; and a purification chamber configured toreceive contaminated carbon dioxide from the collection chamber and toremove contaminants from the contaminated carbon dioxide by apurification material to obtain purified carbon dioxide, thepurification chamber configured to evacuate the purified carbon dioxideand supply it to the bone tissue chamber.

In some embodiments, the system for treating contaminated bone tissuewith purified carbon dioxide also includes a controller and a signaltransmission system functionally interconnecting the controller and thebone tissue chamber, the purified carbon dioxide supply, the collectionchamber and the purification chamber. The controller can also includecomputer readable instructions to cause the controller to effect theevacuation of contaminated carbon dioxide from the bone tissue chamberto the collection chamber, send the contaminated carbon dioxide to thepurification chamber, and dispense the purified carbon dioxide to thebone tissue chamber.

While multiple embodiments are disclosed, still other embodiments of thepresent disclosure will become apparent to those skilled in the art fromthe following detailed description, which shows and describesillustrative embodiments of the disclosure. As will be realized, thevarious embodiments of the present disclosure are capable ofmodifications in various obvious aspects, all without departing from thespirit and scope of the present disclosure. Accordingly, the detaileddescription is to be regarded as illustrative in nature and notrestrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

In part, other aspects, features, benefits and advantages of theembodiments will be apparent with regard to the following description,appended claims and accompanying drawing where:

FIG. 1 illustrates a flow chart of a carbon dioxide method of treatingbone tissue in accordance with one embodiment;

FIG. 2A illustrates a process chart showing the vanishing interfacetension and carbon dioxide pressure for a fiber bone tissue treated inaccordance with one embodiment;

FIG. 2B illustrates a process chart showing the vanishing interfacetension and carbon dioxide pressure for a chip bone tissue treated inaccordance with another embodiment;

FIG. 3 is a simplified schematic of an embodiment of an apparatus orsystem for treating contaminated bone tissue; and

It is to be understood that the figures are not drawn to scale. Further,the relation between objects in a figure may not be to scale, and may infact have a reverse relationship as to size. The figures are intended tobring understanding and clarity to the structure of each object shown,and thus, some features may be exaggerated in order to illustrate aspecific feature of a structure.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of thedisclosure, reference will now be made to certain embodiments andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of thedisclosure is thereby intended, such alterations and furthermodifications in the illustrated methods of decontaminating bone tissue,and such further applications of the principles of the disclosure asdescribed herein being contemplated as would normally occur to oneskilled in the art to which the disclosure relates.

Additionally, unless defined otherwise or apparent from context, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs.

Unless explicitly stated or apparent from context, the following termsare phrases have the definitions provided below:

Definitions

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities of ingredients,percentages or proportions of materials, reaction conditions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about.”Similarly, when values are expressed as approximations, by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment that is +/−10% of the recited value.Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the present disclosure. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Also, as used inthe specification and including the appended claims, the singular forms“a,” “an,” and “the” include the plural, and reference to a particularnumerical value includes at least that particular value, unless thecontext clearly dictates otherwise. Ranges may be expressed herein asfrom “about” or “approximately” one particular value and/or to “about”or “approximately” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of this application are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all subranges subsumedtherein. For example, a range of “1 to 10” includes any and allsubranges between (and including) the minimum value of 1 and the maximumvalue of 10, that is, any and all subranges having a minimum value ofequal to or greater than 1 and a maximum value of equal to or less than10, e.g., 5.5 to 10.

Bioactive agent or bioactive compound is used herein to refer to acompound or entity that alters, inhibits, activates, or otherwiseaffects biological or chemical events. For example, bioactive agents mayinclude, but are not limited to, osteogenic or chondrogenic proteins orpeptides, anti-AIDS substances, anti-cancer substances, antibiotics,immunosuppressants, anti-viral substances, enzyme inhibitors, hormones,neurotoxins, opioids, hypnotics, anti-histamines, lubricants,tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants including channelblockers, miotics and anti-cholinergics, anti-glaucoma compounds,anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand antiadhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, angiogenic factors, anti-secretory factors, anticoagulantsand/or antithrombotic agents, local anesthetics, ophthalmics,prostaglandins, anti-depressants, anti-psychotic substances,anti-emetics, and imaging agents. In certain embodiments, the bioactiveagent is a drug. Bioactive agents further include RNAs, such as siRNA,and osteoclast stimulating factors. In some embodiments, the bioactiveagent may be a factor that stops, removes, or reduces the activity ofbone growth inhibitors. In some embodiments, the bioactive agent is agrowth factor, cytokine, extracellular matrix molecule or a fragment orderivative thereof, for example, a cell attachment sequence such as RGD.A more complete listing of bioactive agents and specific drugs suitablefor use in the present application may be found in “PharmaceuticalSubstances: Syntheses, Patents, Applications” by Axel Kleemann andJurgen Engel, Thieme Medical Publishing, 1999; the “Merck Index: AnEncyclopedia of Chemicals, Drugs, and Biologicals”, edited by SusanBudavari et al., CRC Press, 1996; and the United StatesPharmacopeia-25/National Formulary-20, published by the United StatesPharmacopeia Convention, Inc., Rockville Md., 2001, each of which isincorporated herein by reference.

Biocompatible, as used herein, is intended to describe materials that,upon administration in vivo, do not induce undesirable long-termeffects.

Bone, as used herein, refers to bone that is cortical, cancellous orcortico-cancellous of autogenous, allogenic, xenogenic, or transgenicorigin. Bone is also used in the most general sense and includes alltypes of human or animal bone tissue, including whole bones, bonepieces, bone blocks with attached connective tissues such as ligamentsand tendons, as well as ground bone preparations and grounddemineralized bone preparations.

Demineralized, as used herein, refers to any material generated byremoving mineral material from tissue, for example, bone tissue. Incertain embodiments, the demineralized compositions described hereininclude preparations containing less than 5% calcium. In someembodiments, the demineralized compositions may comprise less than 1%calcium by weight. Partially demineralized bone is intended to refer topreparations with greater than 5% calcium by weight but containing lessthan 100% of the original starting amount of calcium. In someembodiments, partially demineralized comprises 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 and/or 99% ofthe original starting amount of calcium.

In some embodiments, demineralized bone has less than 95% of itsoriginal mineral content. In some embodiments, demineralized bone lessthan 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79,78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61,60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43,42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6and/or 5% of its original content. In some embodiments, “demineralized”is intended to encompass such expressions as “substantiallydemineralized,” “partially demineralized,” “surface demineralized,” and“fully demineralized.” “Partially demineralized” is intended toencompass “surface demineralized.”

In some embodiments, the demineralized bone may be surface demineralizedfrom about 1-99%. In some embodiments, the demineralized bone is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98 and/or 99% surface demineralized. In various embodiments,the demineralized bone may be surface demineralized from about 15-25%.In some embodiments, the demineralized bone is 15, 16, 17, 18, 19, 20,21, 22, 23, 24 and/or 25% surface demineralized.

Demineralized bone activity refers to the osteoinductive activity ofdemineralized bone.

Demineralized bone matrix (DBM), as used herein, refers to any materialgenerated by removing mineral material from bone tissue. In someembodiments, the DBM compositions as used herein include preparationscontaining less than 5% calcium and, in some embodiments, less than 1%calcium by weight. In other embodiments, the DBM compositions comprisepartially demineralized bone (e.g., preparations with greater than 5%calcium by weight but containing less than 100% of the original startingamount of calcium) are also considered within the scope of thisdisclosure.

DBM preparations have been used for many years in orthopedic medicine topromote the formation of bone. For example, DBM has found use in therepair of fractures, in the fusion of vertebrae, in joint replacementsurgery, and in treating bone destruction due to underlying disease suchas a bone tumor. DBM is has been shown to promote bone formation in vivoby osteoconductive and osteoinductive processes. The osteoinductiveeffect of implanted DBM compositions results from the presence of activegrowth factors present on the isolated collagen-based matrix. Thesefactors include members of the TGF-R, IGF, and BMP protein families.Particular examples of osteoinductive factors include TGF-β, IGF-1,IGF-2, BMP-2, BMP-7, parathyroid hormone (PTH), and angiogenic factors.Other osteoinductive factors such as osteocalcin and osteopontin arealso likely to be present in DBM preparations as well. There are alsolikely to be other unnamed or undiscovered osteoinductive factorspresent in DBM.

Lipid, as used herein, refers to any one or more of a group of fats orfat-like substances occurring in humans or animals. The fats or fat-likesubstances are characterized by their insolubility in water andsolubility in organic solvents. Lipid also includes, but is not limitedto, complex lipid, simple lipid, triglycerides, fatty acids,glycerophospholipids (phospholipids), true fats such as esters of fattyacids, glycerol, cerebrosides, waxes, and sterols such as cholesteroland ergosterol. As used herein, lipid also includes lipid-containingorganisms, such as lipid-containing infectious agents. Lipid-containinginfectious agents are defined as any infectious organism or infectiousagent containing lipids. Such lipids may be found, for example, in abacterial cell wall or viral envelope. Lipid-containing organismsinclude but are not limited to eukaryotic and prokaryotic organisms,bacteria, viruses, protozoa, mold, fungi, and other lipid-containingparasites.

Delipidation, as used herein, refers to the process of removing lipidsfrom bone material or from a lipid-containing organisms contained inbone material or tissue.

Contaminants or infectious organisms, as used herein, refer to anylipid-containing infectious organism capable of causing infection. Someinfectious organisms include bacteria, viruses, protozoa, parasites,fungi and mold.

Virus, as used herein, refers to viruses and virus-like particlesincluding enveloped or lipid-coated viruses, and non-enveloped, proteinencased viruses. A “virion” is an individual virus entity or particle.As used herein, the term “inactive” means the virion particle is unableto replicate or infect a host cell.

Osteoconductive, as used herein, refers to the ability of a substance toserve as a template or substance along which bone may grow.

Osteogenic, as used herein, refers to materials containing living cellscapable of differentiation into bone tissue.

Osteoimplant, as used herein, refers to any implant prepared inaccordance with the embodiments described herein and therefore mayinclude expressions such as bone material, bone tissue, bone membrane,bone graft.

Osteoinductive, as used herein, refers to the quality of being able torecruit cells from the host that have the potential to stimulate newbone formation. Any material that can induce the formation of ectopicbone in the soft tissue of an animal is considered osteoinductive. Forexample, most osteoinductive materials induce bone formation in athymicrats when assayed according to the method of Edwards et al.,“Osteoinduction of Human Demineralized Bone: Characterization in a RatModel,” Clinical Orthopaedics & Rel. Res., 357:219-228, December 1998,incorporated herein by reference.

In other instances, osteoinduction is considered to occur throughcellular recruitment and induction of the recruited cells to anosteogenic phenotype. Osteoinductivity score refers to a score rangingfrom 0 to 4 as determined according to the method of Edwards et al.(1998) or an equivalent calibrated test. In the method of Edwards etal., a score of “0” represents no new bone formation; “1” represents1%-25% of implant involved in new bone formation; “2” represents 26-50%of implant involved in new bone formation; “3” represents 51%-75% ofimplant involved in new bone formation; and “4” represents >75% ofimplant involved in new bone formation. In most instances, the score isassessed 28 days after implantation. However, the osteoinductivity scoremay be obtained at earlier time points such as 7, 14, or 21 daysfollowing implantation. In these instances it may be desirable toinclude a normal DBM control such as DBM powder without a carrier, andif possible, a positive control such as BMP. Occasionallyosteoinductivity may also be scored at later time points such as 40, 60,or even 100 days following implantation. Percentage of osteoinductivityrefers to an osteoinductivity score at a given time point expressed as apercentage of activity, of a specified reference score. Osteoinductivitymay be assessed in an athymic rat or in a human. Generally, as discussedherein, an osteoinductive score is assessed based on osteoinductivity inan athymic rat.

Superficially demineralized, as used herein, refers to bone-derivedelements possessing at least about 90 weight percent of their originalinorganic mineral content. In some embodiments, superficiallydemineralized contains at least about 90, 91, 92, 93, 94, 95, 96, 97, 98and/or 99 weight percent of their original inorganic material. Theexpression “partially demineralized” as used herein refers tobone-derived elements possessing from about 8 to about 90 weight percentof their original inorganic mineral content. In some embodiments,partially demineralized contains about 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89 and/or 90 weight percent of their original inorganic mineral content.The expression “fully demineralized” as used herein refers to bonecontaining less than 8% of its original mineral context. In someembodiments, fully demineralized contains about less than 8, 7, 6, 5, 4,3, 2 and/or 1% of its original mineral content.

The expression “average length to average thickness ratio” as applied tothe DBM fibers of the present application means the ratio of the longestaverage dimension of the fiber (average length) to its shortest averagedimension (average thickness). This is also referred to as the “aspectratio” of the fiber.

Fibrous, as used herein, refers to bone elements whose average length toaverage thickness ratio or aspect ratio of the fiber is from about 50:1to about 1000:1. In overall appearance the fibrous bone elements can bedescribed as bone fibers, threads, narrow strips, or thin sheets. Often,where thin sheets are produced, their edges tend to curl up toward eachother. The fibrous bone elements can be substantially linear inappearance or they can be coiled to resemble springs. In someembodiments, the bone fibers are of irregular shapes including, forexample, linear, serpentine or curved shapes. The bone fibers are, insome embodiments, demineralized however some of the original mineralcontent may be retained when desirable for a particular embodiment.

Non-fibrous, as used herein, refers to elements that have an averagewidth substantially larger than the average thickness of the fibrousbone element or aspect ratio of less than from about 50:1 to about1000:1. In some aspects, the non-fibrous bone elements are shaped in asubstantially regular manner or specific configuration, for example,triangular prism, sphere, cube, cylinder and other regular shapes. Bycontrast, particles such as chips, shards, or powders possess irregularor random geometries. It should be understood that some variation indimension will occur in the production of the elements of thisapplication and elements demonstrating such variability in dimension arewithin the scope of this application and are intended to be understoodherein as being within the boundaries established by the expressions“mostly irregular” and “mostly regular”.

Sterilization, as used herein, refers to an act or process using eitherphysical or chemical means for eliminating or inactivating substantiallyall viable organisms, especially micro-organisms, viruses and otherpathogens, associated with a xenograft or bioprosthetic device. As usedherein, “sterilized” includes bone material or bone tissue achieving asterility assurance level of 10⁻⁶ colony forming unit (CFU), asdetermined by FDA (Federal Drug Administration) standards.

Supercritical fluid, as used herein, refers to a substance at atemperature and pressure above its thermodynamic critical point. Underthese conditions, the distinction between gases and liquids does notapply and the substance is described as a fluid. Under these conditions,a supercritical fluid has the ability to diffuse through solids like agas, and dissolve materials like a liquid. Additionally, a supercriticalfluid can readily change in density upon minor changes in temperature orpressure.

Supercritical carbon dioxide, as used herein, refers to carbon dioxide(CO₂) above its thermodynamic critical point (i.e., above criticaltemperature of 31.1° C. and pressure of 1100 psi). Supercritical carbondioxide is an excellent non-polar solvent for many organic compounds. Ithas been likened to a solvent resembling hexane, though with somehydrogen-bonding acceptor capability and some dipole selectivity.Alkenes, alkanes, aromatics, ketones, and alcohols (up to a relativemolecular mass of around 400) dissolve in supercritical carbon dioxide.Very polar molecules such as sugars or amino acids and most inorganicsalts are insoluble. By adjusting the pressure of the fluid, the solventproperties can be adjusted to more “gas-like” or more “liquid-like”,which allows tuning of the solvent properties.

Introduction

The present application is directed to the automated use of criticaland/or supercritical carbon dioxide in preparing decontaminated bonetissue for incorporation into xenografts and bioprosthetic devices.Supercritical fluids such as carbon dioxide can be used to removelipids, contaminants or inactivate infectious agents from the bonetissue under conditions which do not significantly degrade or denaturetissue proteins. The process and apparatus of this application alsoincludes the automated purification of the carbon dioxide that hasbecome contaminated in the process of decontaminating the bone tissue.The contaminated carbon dioxide is collected, purified, liquefied andrecycled for further use in decontaminating the bone tissue contaminatedwith disease causing pathogens, viruses, bacteria, fungi, mildew or amixture thereof. The methods and apparatus for treatment of contaminatedbone tissue described in this disclosure can be a multi batch design.Such an approach is more economical because it allows for the recyclingof purified carbon dioxide rather than the continuous feeding of carbondioxide from an outside source. Recycling carbon dioxide also avoidsreleasing contaminated carbon dioxide reducing not only the overallprocess costs but also improving the environment.

Carbon Dioxide Processing and Apparatus

FIG. 1 illustrates a flow diagram of a carbon dioxide process 100 fordecontamination of bone tissue 102 contaminated with infectiousorganisms such as bacteria, viruses, protozoa, parasites, fungi andmold. Further, in some aspects, the contaminated bone tissue alsoincludes lipids, cells and marrow which could interfere with bonedecontamination and are undesirable in a bone tissue for use to repairbone defects and as bone grafting material. However, it is desirable topreserve beneficial biomaterial in the bone tissue, such as for example,collagen, osteogenic factors, etc. that allow bone growth andintegration of the implant when the bone tissue is implanted into a bonedefect or cavity.

In various embodiments, contaminated bone tissue can have theseundesirable contaminants removed with liquefied, critical orsupercritical carbon dioxide, which extract the contaminants from thebone tissue, and in turn, become contaminated or spent. Contaminated orspent carbon dioxide can then be purified and recycled back for bonetissue decontamination using the carbon dioxide process 100.

Carbon dioxide process 100, in various aspects, includes 5 processingstages. In stage A, the contaminated bone tissue of step 102 ispre-treated by ethanol gradient dehydration in step 104, then packagedin a Tyvek pouch in step 106 or placed in a mesh covering or deliveryfixture in step 108 for further processing in stage B. In stage B, thecontaminated bone tissue of step 110 is treated with liquid carbondioxide (steps 112 and 114) and/or further subjected to critical pointdehydration (CPD) at 45° C. in step 116 and/or treatment withsupercritical carbon dioxide at 105° C. in step 118. The carbon dioxidecontaining contaminants extracted from the treatment of the contaminatedbone tissue can be released at a controlled rate in step 120 for furtherprocessing in stage D, the carbon dioxide recycling stage. In stage B,the contaminated or spent carbon dioxide can be removed (step 122) fromthe carbon dioxide chamber and moved to stages C and D of carbon dioxideprocessing system 100, for further cleaning and/or purifying. In stageC, the contaminated carbon dioxide is cleaned with supercritical fluidin step 124, and sent to recycling stage D for further processing. Someof the cleaned carbon dioxide from step 124 can be and used to cool thecarbon dioxide chamber of step 110 by flushing it with liquid carbondioxide in step 126. In stage D, the carbon dioxide recycling stage, thecontaminated carbon dioxide is collected in step 128, separated from itscontaminants in step 130 and purified in step 132. In the final stage E,the level of purity of the purified carbon dioxide and other processingparameters from stages B and C can be measured by generating processingcharts 134.

In some embodiments, the bone tissue of step 102 may be pre-treated toremove water prior to the critical point drying of stage B. Thus, afterdemineralization, in some aspects, bone tissue samples (in water) may bedehydrated to remove residual water content. Such dehydration may beaccomplished, for example, through a series of graded ethanol solutions(for example, 20%, 50%, 70%, 80%, 90%, 95%, 100% ethanol in deionizedwater) as illustrated in step 104 of FIG. 1. In other embodiments,penetrating the bone tissue with a graded series of ethanol solutions oralcohols may be accomplished in an automated fashion. For example,pressure and vacuum could be used to accelerate penetration into thebone tissue.

In alternative embodiments, other means or procedures for removing water(drying or dehydrating) from the bone tissue may be used. For example,the bone tissue may be washed with other dehydrating liquids such asacetone to remove water, exploiting the complete miscibility of thesetwo fluids. The acetone may then be washed away with high pressureliquid carbon dioxide.

The dehydrated but still contaminated bone tissue can be packaged in adelivery vehicle such as a carrier or covering, for example in a Tyvekpouch as in step 106 or a polymer mesh as in step 108, both illustratedin FIG. 1. For example, a polymer mesh covering is useful because undercontrolled pressure, temperature, treating time, and carbon dioxiderelease, the polymer structures are not affected.

The dehydrated bone tissue is further placed into a carbon dioxidechamber or container used in step 110 for further treatment with carbondioxide. In the carbon dioxide chamber, the dehydrated bone tissue isflushed with a first liquefied carbon dioxide stream in step 112 toextract any ethanol retained from the ethanol gradient dehydration step104. Flushing with liquid carbon dioxide may be done one or more times.For example, in the process illustrated in FIG. 1, flushing with liquidcarbon dioxide is performed a second time at step 114.

In some embodiments, the bone tissue is further subjected to criticalpoint drying which is carried out using carbon dioxide as illustrated atstep 116. The critical point for carbon dioxide is 304.25 K at 7.39 MPaor 31.1° C. at 1072 psi or 31.2° C. and 73.8 bar. To perform criticalpoint drying, the temperature and pressure may continue to be raised,for example to 40° C. with corresponding pressure of 85 bar. In theembodiment illustrated in FIG. 1, the temperature in step 116 at whichcritical point dehydration occurs is raised to 45° C. Thus, in someembodiments, the liquid carbon dioxide is heated until its pressure isat or above the critical point, at which time the pressure can begradually released, allowing the gas to escape and leaving a driedproduct.

In certain embodiments, bone fibers processed using CPD have a BETsurface area from about 1 to about 5 m²/gm, a value 3 or 4 times greaterthan lyophilized bone fibers. In other embodiments, DBM fibers processedusing CPD have a BET area surface from about 40 to about 100 m²/gm, avalue 100 times greater than when DBM fibers are lyophilized.

In a further embodiment, the critical point dried bone tissue mayfurther be treated, or alternatively be treated, with supercriticalcarbon dioxide (carbon dioxide above the critical point) as shown atstep 118 of FIG. 1. Supercritical carbon dioxide may also be useful inviral inactivation. In some embodiments, thus, the bone tissue is placedin a supercritical carbon dioxide chamber and liquid carbon dioxide isintroduced, for example, by an air pump. The temperature is raised to105° C. with corresponding pressure about 485 bar. In alternativeembodiments, other temperatures and/or pressures above the criticalpoint of carbon dioxide may be used. The samples are soaked insupercritical carbon dioxide for a certain time and carbon dioxide isreleased. The resulting bone samples retain surface morphologies, hencesurface area, and osteoinductivity after such treatment.

In yet a further embodiment, monolithic bone is demineralized andparticulated before drying. Accordingly, the bone may be demineralizedin monolithic pieces. The demineralized monolithic pieces may then bemilled in a wet condition and critical point dried, for example usingcarbon dioxide as a medium.

With further reference to FIG. 1, in various embodiments, the carbondioxide carrying the contaminants extracted from the contaminated bonetissue is collected in a used or spent carbon dioxide collection step128. Spent or contaminated carbon dioxide of step 128 is collected intoa collection chamber or container configured to receive contaminatedcarbon dioxide from bone tissue treatment step 110 via the 1^(st) and2^(nd) liquefied carbon dioxide ethanol exchanges steps 112 and 114. Thecollection chamber used in step 128 can also evacuate the contaminatedor spent carbon dioxide to a separation step 130 and/or a purificationstep 132.

The separation step 130 can be performed in a separation chamber usingseveral separation steps. In one embodiment, the contaminated or spentcarbon dioxide is bubbled through water and/or an organic solvent toremove additional amounts of alcohol that may not have been removed inprevious processing steps. In other embodiments, the carbon dioxidebubbled through water and/or organic solvent is further subjected toacid treatment, filtering and liquefaction so that other contaminantssuch as lipids, proteins bacteria and viruses can be removed. Furtherpurification occurs at step 132, wherein the carbon dioxide is passedthrough filters having different pore sizes. Depending on the type ofcontaminants still present in the liquefied carbon dioxide stream,useful purification filters can have pores varying from about 0.2 μm, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 to about 100μm. In various embodiments, the purified carbon dioxide is up to 95.0%,96.0, 97.0, 98.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8 to99.9% free of lipids, disease carrying pathogens, viruses, bacteria,fungi, mildew or mixtures thereof.

The purified liquid carbon dioxide is subsequently recycled to stage Bcarbon dioxide treatment of the contaminated bone tissue, in someaspects, to steps 112 and 114 ethanol extraction steps. The level ofpurification achieved together with other properties of steps 126 and120 can be ascertained from process charts 134 generated in stage E ofthe carbon dioxide processing system 100. In various embodiments,process charts are generated as illustrated in FIGS. 2A and 2B. Theprocess chart illustrated in FIG. 2A measures the vanishing interfacetension (VIT) 210 and carbon dioxide pressure 200 for a treated fiberbone tissue. FIG. 2B depicts VIT measurements 310 and carbon dioxidepressure 300 for a treated chip bone tissue.

FIG. 3 is a simplified schematic of an embodiment of an apparatus orsystem 200 for treating contaminated bone tissue 212. System 200includes a bone tissue chamber 214 configured for holding bone tissuecontaminated with typical bone tissue contaminants, for example diseasecarrying pathogens, bacteria protozoa, viruses, fungi, mildew or amixture thereof. In some embodiments, lipids entrapped in bone tissuecan also be removed with supercritical carbon dioxide. The bone tissuechamber 214, configured for holding contaminated bone tissue 212, isalso configured for receiving purified carbon dioxide from a purifiedcarbon dioxide source 216 through conduit 218 and for evacuating carbondioxide contaminated with the contaminants present in the contaminatedbone tissue through conduit 220 to a collection chamber 222. In someembodiments, the purified carbon dioxide supply 216 can be an externalsource, such as bottled carbon dioxide pressurized containers and/or apurified carbon dioxide reservoir.

The contaminated carbon dioxide from the bone tissue chamber 214 flowsthrough conduit 220 to the collection chamber 222 from which thecontaminated carbon dioxide is evacuated through conduit 224 to a carbondioxide purification chamber or system 226. In the purification chamberor system 226, the contaminated carbon dioxide is purified to removecontaminants to about 99.9% free of lipids, disease causing pathogens,viruses, bacteria, fungi, mildew or a mixture thereof. The carbondioxide purification chamber or system 226 is configured to receivecontaminated carbon dioxide from the collection chamber 222 and toevacuate the purified carbon dioxide through conduit 228 in order torecycle the purified carbon dioxide, where the process can re-start andthe recycled carbon dioxide can be re-used and introduced into the bonetissue chamber 214. If additional carbon dioxide is needed, it can beintroduced from a purified carbon dioxide source 216 through conduit 218and into the bone tissue chamber 214.

The apparatus or system 200 for treating contaminated bone tissue withrecycled purified carbon dioxide can significantly reduce the cost andcomplexity of a supply of purified carbon dioxide only form an externalsource. By recycling the purified carbon dioxide to the bone tissuechamber, the amount and cost of externally delivered carbon dioxide issubstantially reduced. In addition, the automation of the purificationof the carbon dioxide purification treatment for bone fibers and/or bonechips is reduced to a few steps, significantly reducing human error andimproving the reliability of the carbon dioxide treatment process byeliminating potential carbon dioxide non-conformance steps.

In some embodiments, the system for treating contaminated bone tissuewith carbon dioxide further comprises a controller 230 and a signaltransmission system 232 functionally interconnecting the controller 230with all other elements of the system 200 for treatment of contaminatedbone tissue 212, namely, the bone tissue chamber 214, the purifiedcarbon dioxide source 216, the contaminated carbon dioxide collectionchamber 222, the purification chamber of contaminated carbon dioxide 226as well as their interconnecting conduits. For purposes of thisdisclosure, the controller 230 may be embodied as a general purposecomputer, for example a personal computer, or as an industrial typecontroller. The controller 230 can include a CPU 240, a memory 242, aninput/output (I/O) unit, an input unit, a display device, a printingdevice not shown in FIG. 3. The display device, input device andprinting device, need not be integral with the controller 230, but maybe functionally connected to the CPU 240 and/or external to a housingenclosing the CPU 240. The display device may be embodied as any type ofdevice capable of visually communicating with an operator of theapparatus or system 200 for treating contaminated bone tissue, such as,without limitation, one or more annunciators, digital displays, flatpanel displays, CRTs, or other devices capable of providing visualindications relating functioning of the system 200. The input device maybe embodied as buttons, knobs, keypads, touch pads, a computer mouse ortrackball, a keyboard, a microphone and voice recognition software andassociated hardware, or other input device, either integral with thehousing of the controller 230 or external to the housing of thecontroller. The printing device may be embodied as any device capable ofproviding a hard copy output. A sound transducer optionally is providedto effect audible communication relating operation of the controller230. The memory 242 comprises any combination of RAM, ROM, hard drive,flash drive, or CD reader and disc, as required to store instructions tooperate the CPU 240 to effect control of the system 200. The memory 242has stored therein instructions in the form of executable programming.Those skilled in the art will appreciate that software executingprocedures describe herein may be written in any language which can becompiled to operate the CPU 240. It will be understood that controller230 can be linked to a plurality of sensors that detect the level ofcarbon dioxide in each of the chambers to detect low, desired, and highlevels of carbon dioxide as well as temperature, pressure in thechambers and the supply of carbon dioxide to those chambers in theoutlets and inlets can be regulated by the controller 230.

Providing Delipidation

In various embodiments, the carbon dioxide process 100 of FIG. 1 can beutilized for delipidation of fats present in the bone tissue. Easilyavailable and cheap, carbon dioxide is non-toxic, non-corrosive andnon-flammable and, thus well suited for delipidation of bone tissue.Moreover, because carbon dioxide has low viscosity and high diffusioncoefficients, supercritical carbon dioxide can be used to reachcomponents entrapped in bone tissue, such as lipids. The result is thatcarbon dioxide in the supercritical state dissolves the essentiallylipidic organic matter present in the bone tissue easily and virtuallycompletely. The risks to the immune system and of infection fromcontaminated bone tissue are thereby considerably reduced.

In various embodiments, methods are provided for removing at least alipid from bone tissue, the method comprising contacting the bone tissuewith an effective amount of supercritical carbon dioxide therebyobtaining a substantially delipidated bone tissue. In some embodiments,bone tissue subjected to the delipidation methods described herein canbe 99%, 99.5% or 99.9% free of lipids. The treated bone tissue itselfwill contain less than 1%, 0.5% or 0.1% fat on average after treatment,and this amount is evenly distributed.

Terminal Sterilization Using Supercritical Carbon Dioxide

In various aspects, the present application provides methods of removingfrom bone tissue contaminants such as bacteria, viruses, fungi, protozoaand mixtures thereof. The method comprises contacting the bone tissuewith an effective amount of supercritical carbon dioxide sufficient toremove 99.0%, 99.5% or 99.9% of contaminants.

Some bacteria which may be treated by sterilization with supercriticalcarbon dioxide include, but are not limited to the following:Staphylococcus; Streptococcus, including S. pyogenes; Enterococci;Bacillus, including Bacillus anthracis, and Lactobacillus; Listeria;Corynebacterium diphtherias; Gardnerella including G. vaginalis;Nocardia; Streptomyces; Thermoactinomyces vulgaris; Treponema;Camplyobacter; Pseudomonas including P. aeruginosa; Legionella;Neisseria including N. gonorrhoeae and N. meningitides; Flavobacteriumincluding F. meningosepticum and F. odoratum; Brucella; Bordetellaincluding B. pertussis and B. bronchiseptica; Escherichia including E.coli; Klebsiella; Enterobacter; Serratia including S. marcescens and S.liquefaciens; Edwardsiella; Proteus including P. mirabilis and P.vulgaris; Streptobacillus; Rickettsiaceae including R. rickettsii;Chlamydia including C. psittaci and C. trachomatis; Mycobacteriumincluding M. tuberculosis, M. intracellulare, M. fortuitum, M. laprae,M. avium, M. bovis, M. africanum, M. kansasii, M. intracellulare, and M.lepraemurium; and Nocardia, and any other bacteria containing lipid intheir membranes.

Exemplary infectious agents removed from the tissue using the process ofthe application include, viruses, bacteria, mycobacteria, mycoplasma,fungi, prions and constituents thereof. Methods of this application areapplicable to removing viruses of the family of Togaviridae, inparticular of the genus Alphavirus, such as the Hepatitis C virus, andfor preventing their transmission during tissue grafts; for combatingviruses of the family Picorviridae, in particular of the genusEnterovirus, more particularly the Polio Sabin virus, and preventingtheir transmission during tissue grafts; for combating viruses of thefamily Herpesviridae and preventing their transmission during tissuegrafts; for combating viruses of the family Retroviridae, in particularof the genus Lentivirus, more particularly human HIV immunodeficiencyviruses, and preventing their transmission during tissue grafts. Ofparticular interest is the use of the methods of the present applicationto remove prions from contaminated bone tissue.

Embodiments of this application provide methods for inactivatingviruses, especially enveloped or lipid-coated viruses, and nonenveloped,protein encased viruses in proteinaceous products without incurringsubstantial denaturation.

Supercritical carbon dioxide is useful in methods and apparatus forinactivating virus and virus-like particles present in contaminated bonetissues. One embodiment of the present application is directed to amethod of inactivating one or more virions associated with acontaminated bone tissue. In one aspect, the method comprises the stepsof contacting a bone tissue with a critical, near critical orsupercritical carbon dioxide. The critical, near critical orsupercritical carbon dioxide is capable of being received by at leastone virion and upon removal, causes inactivation of the virion. Themethod further comprises the step of removing the critical,supercritical or near critical carbon dioxide from the material and oneor more virions to render one or more virions inactive.

Viral infectious organisms which may be inactivated by the methodsdescribed herein include, but are not limited to the lipid-containingviruses of the following genuses: Alphavirus (alphaviruses), Rubivurus(rubella virus), Flavivirus (Flaviviruses), Pestivirus (mucosal diseaseviruses), (unnamed, hepatitis C virus), Coronavirus, (Coronaviruses),Torovirus, (toroviruses), Arteivirus, (arteriviruses), Paramyxovirus,(Paramyxoviruses), Rubulavirus (rubulaviruses), Morbillivirus(morbillivuruses), Pneumovirinae (the pneumoviruses), Pneumovirus(pneumoviruses), Vesiculovirus (vesiculoviruses), Lyssavirus(lyssaviruses), Ephemerovirus (ephemeroviruses), Cytorhabdovirus (plantrhabdovirus group A), Nucleorhabdovirus (plant rhabdovirus group B),Filovirus (filoviruses), Influenzavirus A, B (influenza A and Bviruses), Influenza virus C (influenza C virus), (unnamed, Thogoto-likeviruses), Bunyavirus (bunyaviruses), Phlebovirus (phleboviruses),Nairovirus (nairoviruses), Hantavirus (hantaviruses), Tospovirus(tospoviruses), Arenavirus (arenaviruses), unnamed mammalian type Bretroviruses, unnamed, mammalian and reptilian type C retroviruses,unnamed type D retroviruses, Lentivirus (lentiviruses), Spumavirus(spumaviruses), Orthohepadnavirus (hepadnaviruses of mammals),Avihepadnavirus (hepadnaviruses of birds), Simplexvirus(simplexviruses), Varicellovirus (varicelloviruses), Betaherpesvirinae(the cytomegaloviruses), Cytomegalovirus (cytomegaloviruses),Muromegalovirus (murine cytomegaloviruses), Roseolovirus (human herpesvirus 6), Gammaherpesvirinae (the lymphocyte-associated herpes viruses),Lymphocryptovirus (Epstein-Bar-like viruses), Rhadinovirus(saimiri-ateles-like herpes viruses), Orthopoxvirus (orthopoxviruses),Parapoxvirus (parapoxviruses), Avipoxvirus (fowl pox viruses),Capripoxvirus (sheeppoxlike viruses), Leporipoxvirus (myxomaviruses),Suipoxvirus (swine-pox viruses), Molluscipoxvirus (molluscum contagiosumviruses), Yatapoxvirus (yabapox and tanapox viruses), Unnamed, Africanswine fever-like viruses, Iridovirus (small iridescent insect viruses),Ranavirus (front iridoviruses), Lymphocystivirus (lymphocystis virusesof fish), Togaviridae, Flaviviridae, Coronaviridae, Enabdoviridae,Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae,Arenaviridae, Retroviridae, Hepadnaviridae, Herpesviridae, Poxyiridae,and any other lipid-containing virus.

These viruses include the following human and animal pathogens: RossRiver virus, fever virus, dengue viruses, Murray Valley encephalitisvirus, tick-borne encephalitis viruses (including European and fareastern tick-borne encephalitis viruses), human coronaviruses 229-E andOC43 and others (causing the common cold, upper respiratory tractinfection, probably pneumonia and possibly gastroenteritis), humanparainfluenza viruses 1 and 3, mumps virus, human parainfluenza viruses2, 4a and 4b, measles virus, human respiratory syncytial virus, rabiesvirus, Marburg virus, Ebola virus, influenza A viruses and influenza Bviruses, Arenaviruss: lymphocytic choriomeningitis (LCM) virus; Lassavirus, human immunodeficiency viruses 1 and 2, or any otherimmunodeficiency virus, hepatitis A virus, hepatitis B virus, hepatitisC virus, Subfamily: human herpes viruses 1 and 2, herpes virus B,Epstein-Barr virus), (smallpox) virus, cowpox virus, molluscumcontagiosum virus.

All protozoa containing lipid, especially in their plasma membranes, areincluded within the scope of the present application. Protozoa that maybe inactivated by treatment of the contaminated bone tissue with carbondioxide at critical and supercritical conditions include, but are notlimited to, the following lipid-containing protozoa: Trypanosoma brucei,Trypanosoma gambiense, Trypanosoma cruzi, Leishmania donovani,Leishmania vianni, Leishmania tropica, Giardia lamblia, Giardiaintestinalis; Trichomonas vaginalis, Entamoeba histolytica, Entamoebacoli, Entamoeba hartmanni, Naegleria species, Acanthamoeba species,Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodiumovale, Toxoplasma gondii, Cryptosporidium parvum, Cryptosporidium muris,Isospora belli, Cyclospora cayetansis, Balantidium species, Babesiabovis, Babesia, microti, Babesia divergens, Encephalitozoonintestinalis, Pleistophora species, Nosema ocularum, Vittaforma corneae,Septata intestinalis, Enterocytozoon, Dientamoeba fragilis, Blastocystisspecies, Sarcocystis species, Pneumocystis carinii, Microsporidiumafricanum, Microsporidium ceylonensis, Eimeria acervulina, Eimeriamaxima, Eimeria tenella and Neospora caninum. It is to be understoodthat the present application is not limited to the protozoa provided inthe list above.

In some embodiments, protozoa treated with methods of the presentapplication is Coccidia, which includes Isospora species,Cryptosporidium species, Cyclospora species, Toxoplasma species,Sarcocystis species, Neospora species, and Eimeria species. Thesecoccidian parasites cause intestinal disease, lymphadenopathy,encephalitis, myocarditis, and pneumonitis.

The terms “protozoal infection” or “infectious disease” mean diseasescaused by protozoal infectious organisms. The diseases include, but arenot limited to, African sleeping sickness, Chagas' disease,Leishmaniasis, Giardiasis, Trichomoniasis, amebiasis, primary amebicencephalitis, granulomatous amebic encephalitis, malaria, Toxoplasmosis,Cryptosporidiosis, Isosporiasis, Cyclosporiasis, Balantidiasis,Babesiosis, microsporidiosis, Dientamoeba fragilis infection,Blastocystis hominis infection, Sarcosporidiosis, pneumonia, andcoccidiosis. A protozoal infection treated with the method of thepresent application is Coccidiosis, which is caused by Isospora species,Cryptosporidium species, Cyclospora species, Toxoplasma species,Sarcocystis species, Neospora species, and Eimeria species. Thesecoccidian parasites cause human intestinal disease, lymphadenopathy,encephalitis, myocarditis, and pneumonitis. These coccidian parasitesalso cause disease in animals, including cattle, dogs, cats, and birds.Avians, and chickens, turkeys and quail in particular, are affected byCoccidiosis, especially by Eimeria species such as E. acervulina, E.maxima, E. necatrix, E. bruneti, E. mitis, E. praecox and E. tenella.

Providing Bone Tissue

The methods of delipidation and decontamination provided by thisapplication apply broadly to bone tissue obtained from any source. Invarious embodiments, in xenogenic implantation in a human subject, bonecan be obtained from animal sources such as cows and pigs. In otherembodiments, in allogenic implantation in a human subject, bone isobtained from human cadavers, following appropriate ethical and legalrequirements. Such human bone tissue is available from a variety oftissue banks.

The bone may comprise cortical bone, cancellous bone, or a combinationthereof. Cancellous bone is available in a range of porosities based onthe location in the body from which the bone is harvested. Highly porouscancellous bone may be harvested from various areas such as the iliaccrest, while less porous bone may be harvested from areas such as thetibial condyle femoral head, and calcaneus. Cortical bone may beobtained from long bones, such as the diaphyseal shaft of the femur andtibia. In certain embodiments, the bone implant comprises cortical bone.

Depending on the desired end-use of the bone composition, the bonetissue may be subjected to mechanical processing. Such processing mayinclude cutting and shaping, in embodiments forming a construct such asa bone pin or disk for implanting. In one embodiment, the presentapplication provides a bone powder. In such an embodiment, the bone isinitially ground to a selected size. In one embodiment, the boneparticulates are less than about 1500 microns in size. In variousembodiments, the bone particles range from about 50 microns to about1000 microns, from about 75 to about 800 microns, or from about 150 toabout 600 microns. Depending on the desired composition, particles maybe of a variety of sizes.

In some embodiments, biological activities of the bone tissue may beincreased. Accordingly, the bone tissue, and compositions formed fromthe bone tissue, may variously be referred to as biologically activeand/or, in some cases, osteoinductive. The biological activities of thebone composition provided herein that may be increased include, but arenot limited to, osteoinductive activity, osteogenic activity,chondrogenic activity, wound healing activity, neurogenic activity,contraction-inducing activity, mitosis-inducing activity,differentiation-inducing activity, chemotactic activity, angiogenic orvasculogenic activity, exocytosis or endocytosis-inducing activity, orother cell or biological activity. It will be appreciated that boneformation processes frequently include a first stage of cartilageformation that creates the basic shape of the bone, which then becomesmineralized (endochondral bone formation). Thus, in many instances,chondrogenesis may be considered an early stage of osteogenesis, thoughof course it may also occur in other contexts.

Providing Bone Particles

The bone-derived tissue may be derived from any vertebrate. In certainembodiments, that the source of the bone tissue can be matched to theeventual recipient of the inventive composition (i.e., the donor andrecipient should, at least, be of the same species). For example, humanbone-derived tissue is typically used in a human subject. In otherembodiments, the bone particles are obtained from bone of xenogenicorigin. Porcine bone and bovine bone are particularly advantageous typesof xenogenic bone tissue that can be used individually or in combinationas sources for the bone particles. Xenogenic bone tissue may be combinedwith allogenic or autogenous bone.

Methods for the preparation of bone particles are known in the art. Boneparticles can be formed by milling whole bone to produce fibers,chipping whole bone, cutting whole bone, fracturing whole bone in liquidnitrogen, or otherwise disintegrating the bone tissue. In certainembodiments, particles are sieved to produce particles of a specificsize range. Bone particles may be of any shape or size. Exemplary shapesinclude spheroidal, plates, fibers, cuboidal, sheets, rods, oval,strings, elongated particles, wedges, discs, rectangular, polyhedral. Insome embodiments, bone particles may be between about 10 microns andabout 1000 microns in diameter or more. In some embodiments, particlesmay be between about 20 microns and about 800 microns in diameter ormore. In certain embodiments, the particles range in size fromapproximately 100 microns in diameter to approximately 500 microns indiameter. In certain embodiments, the particles range in size fromapproximately 300 microns in diameter to approximately 800 microns indiameter. As for irregularly shaped particles, the recited dimensionranges may represent the length of the greatest or smallest dimension ofthe particle.

In certain embodiments, the bone-derived particles are used “as is” inpreparing the inventive composites. In other embodiments, thebone-derived particles are modified before composite preparation. Thus,for example, bone particles suitable for use in the methods of thepresent application can be demineralized, non-demineralized,mineralized/deorganified, or anorganic bone particles.

Providing Demineralized Bone Tissue

Following shaving, milling or other technique whereby they are obtained,the bone tissue is subjected to demineralization in order to reduce itsinorganic content to a very low level, in some embodiments, to not morethan about 5% by weight of residual calcium and, in other aspects, tonot more than about 1% by weight residual calcium.

Demineralization of the bone tissue ordinarily results in itscontraction to some extent. Bone used in the methods described hereinmay be autograft, allograft, or xenograft. In various embodiments, thebone may be cortical bone, cancellous bone, or cortico-cancellous bone.While specific discussion is made herein to demineralized bone tissue,bone tissue treated in accordance with the teachings herein may benon-demineralized, demineralized, partially demineralized, or surfacedemineralized. The following discussion applies to demineralized,partially demineralized, and surface demineralized bone tissue. In oneembodiment, the demineralized bone is sourced from bovine or human bone.In another embodiment, demineralized bone is sourced from human bone. Inone embodiment, the demineralized bone is sourced from the patient's ownbone (autogenous bone). In another embodiment, the demineralized bone issourced from a different animal (including a cadaver) of the samespecies (allograft bone).

Any suitable manner of demineralizing the bone may be used.Demineralization of the bone tissue can be conducted in accordance withknown conventional procedures. For example, in a demineralizationprocedure, the bone tissue useful for the methods of this disclosure issubjected to an acid demineralization step that is followed by adefatting/disinfecting step. The bone tissue is immersed in acid overtime to effect its demineralization. Acids which can be employed in thisstep include inorganic acids such as hydrochloric acid and organic acidssuch as peracetic acid, acetic acid, citric acid, or propionic acid. Thedepth of demineralization into the bone surface can be controlled byadjusting the treatment time, temperature of the demineralizingsolution, concentration of the demineralizing solution, agitationintensity during treatment, and other applied forces such as vacuum,centrifuge, pressure, and other factors such as known to those skilledin the art. The defatting/disinfecting step can be accomplished by themethod of delipidation/terminal sterilization utilizing contacting thebone tissue with supercritical fluid as described in this application.Thus, in various embodiments, the bone tissue may be fullydemineralized, partially demineralized, or surface demineralized.

In other embodiments, the delipidation/terminal sterilization methods ofthe present application can also be used as an additional viralinactivation method following a conventional/defatting disinfectingstep.

After acid treatment, the bone tissue is rinsed with sterile water forinjection, buffered with a buffering agent to a final predetermined pHand then finally rinsed with water for injection to remove residualamounts of acid and buffering agent or washed with water to removeresidual acid and thereby raise the pH. Following demineralization, thebone tissue is immersed in solution to effect its defatting. Further, inaccordance with this application, the demineralized bone tissue can beused immediately for preparation of the implant composition or it can bestored under aseptic conditions, advantageously in a critical pointdried state prior to such preparation. In an embodiment, the bone tissuecan retain some of its original mineral content such that thecomposition is rendered capable of being imaged utilizing radiographictechniques.

The bone tissue may be particulated. If the bone tissue isdemineralized, the bone may be particulated before, during or afterdemineralization. As previously discussed, in some embodiments, the bonetissue may be monolithic and may not be particulated. Accordingly, whilespecific discussion is given to particulating bone, the methodsdisclosed herein and the nanoscale textured surfaces disclosed hereinmay be used with monolithic bones or implants, including, for example,surface demineralized implants or fully demineralized cortical boneimplants.

The bone tissue may be milled and ground or otherwise processed intoparticles of an appropriate size before or after demineralization. Theparticles may be particulate or fibrous. The terms milling or grindingare not intended to be limited to production of particles of a specifictype and may refer to production of particulate or fibrous particles. Incertain embodiments, the particle size may be greater than 75 microns,such as ranging from about 100 to about 3000 microns, or from about 200to about 2000 microns. After grinding, the bone particles may be sievedto select those particles of a desired size. In certain embodiments, theparticles may be sieved though a 50 micron sieve, a 75 micron sieve, ora 100 micron sieve.

In yet a further embodiment, monolithic bone tissue is demineralized andparticulated before drying. Accordingly, the bone tissue may bedemineralized in monolithic pieces. The demineralized monolithic piecesmay then be milled in a wet condition and critical point dried, forexample using carbon dioxide as a medium.

In yet a further embodiment, monolithic bone is demineralized and driedbefore particulating (if done). Accordingly, the bone may bedemineralized in monolithic pieces. The DBM is pressed in a wetcondition and then critical point dried, for example using carbondioxide as a medium. In alternatives of this embodiment, thedemineralized and dried monolithic bone is not particulated and isprocessed as a monolithic implant.

Providing Demineralized Bone Matrix

In various embodiments, the process of this application can be used todecontaminate bone matrix compositions which comprise fibers. DBMincludes the collagen matrix of the bone together with acid insolubleproteins including bone morphogenetic proteins (BMPs) and other growthfactors. It can be formulated for use as granules, gels, sponge materialor putty and can be freeze-dried for storage. Sterilization proceduresused to protect from disease transmission may reduce the activity ofbeneficial growth factors in the DBM. DBM provides an initialosteoconductive matrix and exhibits a degree of osteoinductivepotential, inducing the infiltration and differentiation ofosteoprogenitor cells from the surrounding tissues.

DBM preparations have been used for many years in orthopedic medicine topromote the formation of bone. For example, DBM has found use in therepair of fractures, in the fusion of vertebrae, in joint replacementsurgery, and in treating bone destruction due to underlying disease suchas rheumatoid arthritis. DBM is thought to promote bone formation invivo by osteoconductive and osteoinductive processes. The osteoinductiveeffect of implanted DBM compositions is thought to result from thepresence of active growth factors present on the isolated collagen-basedmatrix. These factors include members of the TGF-β, IGF, and BMP proteinfamilies. Particular examples of osteoinductive factors include TGF-β,IGF-1, IGF-2, BMP-2, BMP-7, parathyroid hormone (PTH), and angiogenicfactors. Other osteoinductive factors such as osteocalcin andosteopontin are also likely to be present in DBM preparations as well.There are also likely to be other unnamed or undiscovered osteoinductivefactors present in DBM.

In various embodiments, the DBM for use in the methods described in thisapplication is prepared from elongated bone fibers which have beensubjected to critical point drying. The elongated bone fibers employedin this application are generally characterized as having relativelyhigh average length to average width ratios, also known as the aspectratio. In various embodiments, the aspect ratio of the elongated bonefibers is at least from about 50:1 to about at least about 1000:1. Suchelongated bone fibers can be readily obtained by any one of severalmethods, for example, by milling or shaving the surface of an entirebone or relatively large section of bone.

In other embodiments, the length of the fibers can be at least about 3.5cm and average width from about 20 mm to about 1 cm. In variousembodiments, the average length of the elongated fibers can be fromabout 3.5 cm to about 6.0 cm and the average width from about 20 mm toabout 1 cm. In other embodiments, the elongated fibers can have anaverage length be from about 4.0 cm to about 6.0 cm and an average widthfrom about 20 mm to about 1 cm.

In yet other embodiments, the diameter or average width of the elongatedfibers is, for example, not more than about 1.00 cm, not more than 0.5cm or not more than about 0.01 cm. In still other embodiments, thediameter or average width of the fibers can be from about 0.01 cm toabout 0.4 cm or from about 0.02 cm to about 0.3 cm.

In another embodiment, the aspect ratio of the fibers can be from about50:1 to about 950:1, from about 50:1 to about 750:1, from about 50:1 toabout 500:1, from about 50:1 to about 250:1; or from about 50:1 to about100:1. Fibers according to this disclosure can advantageously have anaspect ratio from about 50:1 to about 1000:1, from about 50:1 to about950:1, from about 50:1 to about 750:1, from about 50:1 to about 600:1,from about 50:1 to about 350:1, from about 50:1 to about 200:1, fromabout 50:1 to about 100:1, or from about 50:1 to about 75:1.

To prepare the osteogenic DBM, a quantity of fibers is combined with abiocompatible carrier to provide a demineralized bone tissue.

Providing a Carrier

Generally, materials for the carrier may be biocompatible in vivo andoptionally biodegradable. In some uses, the carrier acts as a temporaryscaffold until replaced completely by new bone. Suitable carriers can beany number of compounds and/or polymers, such as polymer sugars,proteins, long chain hydrophilic block copolymers, reverse phase blockcopolymers, hyaluronic acid, polyuronic acid, mucopolysaccharide,proteoglycan, polyoxyethylene, surfactants, including the pluronicsseries of nonionic surfactants, and peptide thickener. Suggested classesof biocompatible fluid carrier would include polyhydroxy compound,polyhydroxy ester, fatty alcohol, fatty alcohol ester, fatty acid, fattyacid ester, liquid silicone, combinations thereof, and the like.Settable materials may be used, and they may set up either in situ, orprior to implantation. The bone fibers and carrier (or delivery orsupport system) together form an osteoimplant useful in clinicalapplications.

Examples of suitable biocompatible fluid carrier include, but are notlimited to:

(i) Polyhydroxy compound, for example, such classes of compounds as theacyclic polyhydric alcohols, non-reducing sugars, sugar alcohols, sugaracids, monosaccharides, disaccharides, water-soluble or waterdispersible oligosaccharides, polysaccharides and known derivatives ofthe foregoing. Specific polyhydroxy compounds include, 1,2-propanediol,glycerol, 1,4,-butylene glycol trimethylolethane, trimethylolpropane,erythritol, pentaerythritol, ethylene glycols, diethylene glycol,triethylene glycol, tetraethylene glycol, propylene glycol, dipropyleneglycol; polyoxyethylene-polyoxypropylene copolymer, for example, of thetype known and commercially available under the trade names Pluronic andEmkalyx; polyoxyethylene-polyoxypropylene block copolymer, for example,of the type known and commercially available under the trade namePoloxamer; alkylphenolhydroxypolyoxyethylene, for example, of the typeknown and commercially available under the trade name Triton,polyoxyalkylene glycols such as the polyethylene glycols, xylitol,sorbitol, mannitol, dulcitol, arabinose, xylose, ribose, adonitol,arabitol, inositol, fructose, galactose, glucose, mannose, sorbose,sucrose, maltose, lactose, maltitol, lactitol, stachyose, maltopentaose,cyclomaltohexaose, carrageenan, agar, dextran, alginic acid, guar gum,gum tragacanth, locust bean gum, gum arabic, xanthan gum, amylose,mixtures of any of the foregoing, and the like.

(ii) Polyhydroxy ester, for example, liquid and solid monoesters anddiesters of glycerol can be used to good effect, the solid esters beingdissolved up in a suitable vehicle, for example, propylene glycol,glycerol, polyethylene glycol of 200-1000 molecular weight. Liquidglycerol esters include monacetin and diacetin and solid glycerol estersinclude such fatty acid monoesters of glycerol as glycerol monolaurate,glyceryl monopalmitate, glyceryl monostearate. In various embodiments,the carrier herein comprises glyceryl monolaurate dissolved in glycerolor a 4:1 to 1:4 weight mixtures of glycerol and propylene glycol, poly(oxyalkylene) glycol ester, and the like.

(iii) Fatty alcohol, for example primary alcohols, usually straightchain having from 6 to 13 carbon atoms, including caproic alcohol,caprylic alcohol, undecyl alcohol, lauryl alcohol, and tridecanol.

(iv) Fatty alcohol ester, for example, ethyl hexyl palmitate, isodecylneopentate, octadodecyl benzoate, diethyl hexyl maleate, and the like.

(v) Fatty acid having from 6 to 11 carbon atoms, for example, hexanoicacid, heptanoic acid, octanoic acid, decanoic acid and undecanoic acid.

(vi) Fatty acid ester, for example, polyoxyethylene-sorbitan-fatty acidesters, for example, mono- and tri-lauryl, palmityl, stearyl, and oleylesters including of the type available under the trade name Tween fromImperial Chemical Industries; polyoxyethylene fatty acid estersincluding polyoxyethylene stearic acid esters of the type known andcommercially available under the trade name Myrj; propylene glycol mono-and di-fatty acid esters such as propylene glycol dicaprylate; propyleneglycol dilaurate, propylene glycol hydroxy stearate, propylene glycolisostearate, propylene glycol laureate, propylene glycol ricinoleate,propylene glycol stearate, and propylene glycol caprylic-capric aciddiester available under the trade name Miglyol; mono-, di-, andmono/di-glycerides, such as the esterification products of caprylic orcaproic acid with glycerol, for example, of the type known andcommercially available under the trade name IMWITOR®; sorbitan fattyacid esters, or of the type known and commercially available under thetrade name Span, including sorbitan-monolauryl,-monopalmityl,-monostearyl, -tristearyl, -monooleyl and triolcylesters;monoglycerides, for example, glycerol monooleate, glycerol monopalmitateand glycerol monostearate, for example, as known and commerciallyavailable under the trade names Myvatex, Myvaplex and Myverol, andacetylated, for example, mono- and di-acetylated monoglycerides, forexample, as known and commercially available under the trade nameMyvacet; isobutyl tallowate, n-butylstearate, n-butyl oleate, andn-propyl oleate.

(vii) Liquid silicone, for example, polyalkyl siloxanes such aspolymethyl siloxane and poly (dimethyl siloxane) and polyalkylarylsiloxane.

In some embodiments of the implantable composition of this application,the liquid carrier is a liquid polyhydroxy compound, liquid polyhydroxycompound derivative, liquid solution of solid polyhydroxy compound,liquid solution of solid polyhydroxy compound derivative or combinationsthereof. If necessary or desirable, in some embodiments, the liquidcarrier can be dissolved or diluted with an appropriate solvent suchthat when combined with the demineralized bone fibers described herein acomposition capable of being shaped or packed into a coherent mass whichretains its shape and volume over the relatively long term, until thebone formation and remodeling process is completed, is provided. Thus,the polyhydroxy compound or polyhydroxy derivatives can be a liquid inthe pure or highly concentrated state at ambient temperature, from about15° C. to about 50° C., or it can be a solid or semi-solid at thistemperature in which case it becomes necessary to dissolve the materialin a solvent such as water, physiological saline, ethanol, glycerol,glucose, propylene glycol, polyethylene glycol of from 200-1000molecular weight, or polyvinyl alcohol. In other embodiments, the liquidcarrier can be made up of one or more liquid polyhydroxy compounds orderivatives in solution with one or more solid polyhdroxy compounds orderivatives.

The osteoinductive or biologically active composition may be configuredto be moldable, extrudable, or substantially solid. The osteoinductiveor biologically active composition may be configured to substantiallyretain its shape in water for a period of time. The osteoinductive orbiologically active composition may form an osteoimplant useful inclinical applications. Suitable carriers may include surfacedemineralized bone; mineralized bone; nondemineralized cancellousscaffolds; demineralized cancellous scaffolds; cancellous chips;particulate, demineralized, guanidine extracted, species-specific(allogenic) bone; specially treated particulate, protein extracted,demineralized, xenogenic bone; collagen; synthetic hydroxyapatites;synthetic calcium phosphate materials; tricalcium phosphate, sinteredhydroxyapatite, settable hydroxyapatite; polylactide polymers;polyglycolide polymers, polylactide-co-glycolide copolymers; tyrosinepolycarbonate; calcium sulfate; collagen sheets; settable calciumphosphate; polymeric cements; settable poly vinyl alcohols,polyurethanes; resorbable polymers; and other large polymers; liquidsettable polymers; and other biocompatible settable materials. Thecarrier may further comprise a polyol (including glycerol or otherpolyhydroxy compound), a polysaccharide (including starches), a hydrogel(including alginate, chitosan, dextran, pluronics,N,O-carboxymethylchitosan glucosamine (NOCC)), hydrolyzed cellulose, ora polymer (including polyethylene glycol). In embodiments whereinchitosan is used as a carrier, the chitosan may be dissolved using knownmethods including in water, in mildly acidic aqueous solutions, inacidic solutions.

The carrier may further comprise a hydrogel such as hyaluronic acid,dextran, pluronic block copolymers of polyethylene oxide andpolypropylene, and others. Suitable polyhodroxy compounds include suchclasses of compounds as acyclic polyhydric alcohols, non-reducingsugars, sugar alcohols, sugar acids, monosaccharides, disaccharides,water-soluble or water dispersible oligosaccharides, polysaccharides andknown derivatives of the foregoing. An example carrier comprisesglyceryl monolaurate dissolved in glycerol or a 4:1 to 1:4 weightmixture of glycerol and propylene glycol. Settable materials may beused, and they may set up either in situ, or prior to implantation.Optionally, xenogenic bone powder carriers also may be treated withproteases such as trypsin. Xenogenic carriers may be treated with one ormore fibril modifying agents to increase the intraparticle intrusionvolume (porosity) and surface area. Useful agents include solvents suchas dichloromethane, trichloroacetic acid, acetonitrile and acids such astrifluoroacetic acid and hydrogen fluoride. The choice of carrier maydepend on the desired characteristics of the composition. In someembodiments, a lubricant, such as water, glycerol, or polyethyleneglycol may be added.

Any suitable shape, size, and porosity of carrier may be used. In someembodiments, the carrier may be settable and/or injectable. Such carriermay be, for example, a polymeric cement, a suitable settable calciumphosphate, a settable poly vinyl alcohol, a polyurethane, or a liquidsettable polymer. Hydrogel carriers may additionally impart improvedspatial properties, such as handling and packing properties, to theosteoconductive composition. An injectable carrier may be desirablewhere the composition is used with a containment device. In addition,selected materials must be biocompatible in vivo and optionallybiodegradable. In some uses, the carrier acts as a temporary scaffolduntil replaced by new bone. Polylactic acid (PLA), polyglycolic acid(PGA), and various combinations have different dissolution rates invivo. In bone, the dissolution rates can vary according to whether thecomposition is placed in cortical or trabecular bone.

In certain embodiments, the carrier may comprise a shape-retaining solidmade of loosely adhered particulate material with collagen. It mayalternatively comprise a molded, porous solid, a monolithic solid, or anaggregate of close-packed particles held in place by surrounding tissue.Masticated muscle or other tissue may also be used. Large allogenic boneimplants may act as a carrier, for example where their marrow cavitiesare cleaned and packed with DBM and, optionally, the osteoinductivefactors.

In various embodiments, the carrier comprises an osteoinductive materialsuch as a mineralized particulated material, osteoinductive growthfactors, or partially demineralized bone. The mineralized particulatedmaterial may be TCP, hydroxyapatite, mineral recovered from bone,cancellous chips, cortical chips, surface demineralized bone, or othermaterial. The osteoinductive material may be combined with a furthercarrier such as starch or glycerol. Accordingly, in some embodiments,the bone tissue may act as a carrier for the tissue-derived extract.

Where, in a particular implantable composition, the fibrous and/ornon-fibrous elements exhibit a tendency to quickly or prematurelyseparate from the carrier component or to otherwise settle out from thecomposition such that application of a fairly homogeneous composition isrendered difficult or inconvenient, it can be advantageous to includewithin the composition an optional substance whose thixotropiccharacteristics prevent or reduce this tendency. Thus, for example,where the carrier component is glycerol and separation of fibrous and/ornon-fibrous bone elements occurs to an excessive extent where aparticular application is concerned, a thixotropic agent such as asolution of polyvinyl alcohol, polyvinylpyrrolidone, cellulosic estersuch as hydroxypropyl methylcellulose, carboxyl methylcellulose, pectin,food-grade texturizing agent, gelatin, dextran, collagen, starch,hydrolyzed polyacrylonitrile, hydrolyzed polyacrylamide, polyelectrolytesuch as polyacrylic acid salt, hydrogels, chitosan, other materials thatcan suspend the fibrous and/or non-fibrous elements, can be combinedwith the carrier in an amount sufficient to significantly improve thesuspension-keeping characteristics of the composition.

Preparing a DBM Composition

To prepare a DBM composition according to one or more embodiments ofthis application, a quantity of demineralized bone fibers prepared asdescribed above is combined with water or any other appropriate,biocompatible liquid to form a smooth, flowable, cohesive paste. Theresultant implantable composition may be molded or injected into anydesired shape and retains its shape, even when submersed in water,saline, or other aqueous solution. An additional benefit of the DBMfibers is that the resultant paste is injectable through an 18-gaugeneedle.

The liquid may be any biocompatible liquid, including water, salinesolution, buffered solutions, serum, bone marrow aspirant, blood,platelet-rich plasma and the like and combinations thereof. Somebiocompatible liquids suitable for use with the short DBM fibers, suchas serum, bone marrow aspirant and blood, additionally containosteoinductive factors that will promote bone growth at the site towhich the composition is applied.

Providing Optional Additives

If desired, the fibrous and/or non-fibrous bone tissue of thisapplication can be modified in one or more ways. In various embodiments,any of a variety of medically and/or surgically useful optionalsubstances can be incorporated in, or associated with, the bone elementsbefore, during, or after preparation of the implantable composition.Thus, in some embodiments, one or more of such substances can beintroduced into the bone tissue, for example, by soaking or immersingthe bone tissue in a solution or dispersion of the desired substance(s),by adding the substance(s) to the carrier component of the implantablecomposition or by adding the substance(s) directly to the implantablecomposition.

Medically/surgically useful substances which can be readily combinedwith the bone fibers, fluid carrier and/or implantable composition ofthis application include, for example, collagen, insoluble collagenderivatives, hydroxyapatite, and soluble solids and/or liquids dissolvedtherein, for example, antiviricides, particularly those effectiveagainst HIV and hepatitis; antimicrobials and/or antibiotics such aserythromycin, bacitracin, neomycin, penicillin, polymyxin B,tetracyclines, viomycin, chloromycetin and streptomycins, cefazolin,ampicillin, azactam, tobramycin, clindamycin and gentamycin; aminoacids, peptides, vitamins, inorganic elements, inorganic compounds,cofactors for protein synthesis, hormones; endocrine tissue or tissuefragments; synthesizers; enzymes such as collagenase, peptidases,oxidases; polymer cell scaffolds with paraenchymal cells; angiogenicdrugs and polymeric carriers containing such drugs; collagen lattices;biocompatible surface active agents; antigenic agents; cytoskeletalagents; cartilage fragments, living cells such as chondrocytes, bonemarrow cells, mesenchymal stem cells, natural extracts, tissuetransplants, bioadhesives, bone morphogenetic proteins (BMPs),transforming growth factor (TGF-beta), insulin-like growth factor(IGF-1) (IGF-2), platelet derived growth factor (PDGF), fibroblastgrowth factors (FGF), vascular endothelial growth factor (VEGF),angiogenic agents, bone promoters, cytokines, interleukins, geneticmaterial, genes encoding bone promoting action, cells containing genesencoding bone promoting action; growth hormones such as somatotropin;bone digestors; antitumor agents; fibronectin; cellular attractants andattachment agents; immunosuppressants; permeation enhancers, forexample, fatty acid esters such as laureate, myristate and stearatemonesters of polyethylene glycol, surface active agents, enaminederivatives, α-keto aldehydes; nucleic acids; epidermal growth factor(EGF); all collagen types (not just type 1); non-collagenous proteinssuch as osteopontin, osteonectine, bone sialo proteins, vitronectin,thrombospondin, proteoglycans, decorin, biglycan, aggrecan, versican,tenascin, matrix gla protein hyaluronan; soluble and insolublecomponents of the immune system, soluble and insoluble receptorsincluding truncated forms, soluble, insoluble and cell surface boundligands including truncated forms; chemokines, bioactive compounds thatare endocytosed; compounds capable of altering the membrane potential ofcells, compounds capable of altering the monovalent and divalentcation/anion channels of cells; bone resorption inhibitors andstimulators; angiogenic and mitogenic factors; bioactive factors thatinhibit and stimulate second messenger molecules; integrin adhesionmolecules; clotting factors; externally expanded autograft or xenograftcells and any combinations thereof. The amounts of such optionally addedsubstances can vary widely with optimum levels being readily determinedin a specific case by routine experimentation.

The demineralized bone tissue produced with the bone fibers prepared bydelipidation/terminal sterilization described herein may comprise anumber of materials in combination, some or all of which may be in theform of fibers and/or particles. The matrix may comprise calciumphosphates. Driessens, et al. “Calcium phosphate bone cements,” Wise, D.L., Ed., Encyclopedic Handbook of Biomaterials and Bioengineering, PartB, Applications New York: Marcel Decker; Elliott, Structure andChemistry of the Apatites and Other Calcium Phosphates Elsevier,Amsterdam, 1994, each of which is incorporated by reference. Calciumphosphate matrices include, but are not limited to, dicalcium phosphatedihydrate, monetite, tricalcium phosphate, tetracalcium phosphate,hydroxyapatite, nanocrystalline hydroxyapatite, poorly crystallinehydroxyapatite, substituted hydroxyapatite, and calcium deficienthydroxyapatites. In some embodiments, the bone fibers may be added to acarrier.

In some embodiments, the demineralized bone may be further treated toaffect properties of the bone. For example, the DBM may be treated todisrupt the collagen structure of the DBM. Such treatment may comprisecollagenase treatment, heat treatment, mechanical treatment, or other.While demineralized bone is specifically discussed herein, in someembodiments, the teachings herein may be applied to non-demineralizedbone, to partially demineralized bone, or to surface demineralized bone.

In accordance with various embodiments, the bone tissue provided hereinmay be used with growth factors, extracts, peptide hormones, or otheradditives to increase the osteoinductive capacity or that otherwiseencourage cell or biological activity of the bone tissue or to impartother benefits to the bone tissue. It will be appreciated that theamount of additive used will vary depending upon the type of additive,the specific activity of the particular additive preparation employed,and the intended use of the composition. The desired amount is readilydeterminable by the user.

Any of a variety of medically and/or surgically useful optionalsubstances can be incorporated in, or associated with, theosteoinductive factors either before, during, or after preparation ofthe osteoinductive or biologically active composition. Thus, forexample, when demineralized bone fibers prepared bydelipidation/terminal sterilization described herein are used to formthe material, one or more of such substances may be introduced into thedemineralized bone fibers, by soaking or immersing these bone fibers ina solution or dispersion of the desired substance(s).

In one embodiment, a tissue-derived extract may be added to the bonetissue. U.S. Pat. No. 8,357,384 discloses such extracts and addition ofsuch extracts to DBM and is incorporated herein by reference. Forexample, a tissue-derived extract or partially demineralized bone may beadded to the bone tissue. The extract may be derived from any suitabletissue, such as bone, bladder, kidney, brain, skin, or connectivetissue. Further, the extract may be derived in any suitable manner. Theextract may be allogeneic, autogeneic, xenogeneic, or transgenic. Inembodiments wherein the extract is bone-derived, the bone may becortical, cancellous, or corticocancellous and may be demineralized,partially demineralized, or mineralized. In some embodiments, theextract may comprise demineralized bone, partially demineralized bone,mineral derived from bone, or collagen derived from bone. In someembodiments, the tissue-derived extract may be a protein extract.

Bone regeneration involves a multitude of cells, for example, cartilage,fibroblasts, endothelial cells besides osteoblasts. Accordingly, thebone tissue composition may be used to deliver stem cells, which offersthe potential to give rise to different types of cells in the bonerepair process. In one embodiment, the bone tissue composition furthercomprises a cell such as an osteogenic cell or a stem cell.

In various embodiments, the additive may comprise radiopaque substances,angiogenesis promoting materials, bioactive agents, osteoinducingagents, or other. Such materials would include without limitation bariumsulfate, iodine-containing compounds, titanium and mineralized bone.

In certain embodiments, the additive is adsorbed to or otherwiseassociated with the bone tissue. The additive may be associated with thebone tissue through specific or non-specific interactions, or covalentor noncovalent interactions. Examples of specific interactions includethose between a ligand and a receptor, an epitope or an antibody.Examples of nonspecific interactions include hydrophobic interactions,electrostatic interactions, magnetic interactions, dipole interactions,van der Waals interactions, or hydrogen bonding. In certain embodiments,the additive is attached to the bone tissue composition, for example, tothe carrier, using a linker so that the additive is free to associatewith its receptor or site of action in vivo. In other embodiments theadditive is either covalently or non-covalently attached to the carrier.In certain embodiments, the additive may be attached to a chemicalcompound such as a peptide that is recognized by the carrier. In anotherembodiment, the additive is attached to an antibody, or fragmentthereof, that recognizes an epitope found within the carrier. In certainembodiments at least additives are attached to the osteoimplant. Inother embodiments at least three additives are attached to theosteoinductive or biologically active composition. An additive may beprovided within the osteoinductive or biologically active composition ina sustained release format. For example, the additive may beencapsulated within biodegradable polymer nanospheres, or microspheres.

Flow additives according to this application can include, but are notlimited to, small molecule organic compounds, polymeric/oligomericmaterials, and solutions thereof. In some embodiments, when added to theimplantable composition containing the bone fibers the viscosity thereofshould be sufficiently changed to allow flow through a syringe needle ofabout 8-gauge or greater (greater number gauges of syringe needles havesmaller diameters, thus requiring lower threshold viscosity throughwhich they may flow), in some aspects, of about 12-gauge or greater, forexample of about 14-gauge or greater, of about 15-gauge or greater, orof about 18-gauge or greater. Sufficient flow can be understood, interms of syringe needles, to result in an injection force of not morethan 50 pounds, and in some aspects, not more than 40 pounds. In anotherembodiment, the flow additive modifies the viscosity of the compositionto which it is added such that the composition is capable of flowingthrough a syringe needle having a gauge size from about 8 to about 18,alternately from about 8 to about 15, from about 12 to about 18, or fromabout 12 to about 15.

When present, the amount of flow additive that can be added to thecomposition can be from about 0.01% to about 1.5% by weight of the fibercomposition from about 0.1% to about 1% by weight, or from about 0.05%to about 1% by weight. In an alternate embodiment, the amount of flowadditive can be from about 1.5% to about 5% by weight of the fibercomposition. In an embodiment, the flow additive, when used, is presentin an amount of about 0.5% by weight of the composition.

Suitable examples of flow additives can include, but are in no waylimited to, hyaluronic acid; hyaluronate salts such as sodium,potassium, lithium, or the like, or a combination thereof; alginatesalts such as sodium, potassium, lithium, or the like; starch compounds,which can be present in its natural form, in a destructured form, or inany number of chemically modified derivative forms (for example,alkyoxylated derivatives, esterified derivatives, ionically modifiedstarches, oxidized starches, grafted starches, crosslinked starches, orthe like, or combinations thereof); saturated, monounsaturated, and/orpolyunsaturated oils, such as those extracted or isolated from plantand/or animal sources, including, but not limited to, sunflower,safflower, peanut, castor bean, sesame, coconut, soybean, corn, canola,olive, vegetable, palmitins, stearins, oleins, and the like, orderivatives or combinations thereof, as naturally extracted, assynthesized, or as modified or processed in some way, partially or fullyhydrogenated, partially or fully dehydrogenated, partially or fullysaponified, partially or fully acidified, partially halogenated, or thelike; a wax including, but not limited to, hydrocarbon waxes (forexample, polyolefin waxes, such as polyethylene wax, polypropylene wax,and the like, or copolymers thereof), oligoester waxes, monoester waxes,oligoether waxes, monoether waxes, and the like, or combinationsthereof, as naturally extracted, as synthesized, or as modified orprocessed in some way, partially or fully hydrogenated, partially orfully dehydrogenated, partially or fully saponified, partially or fullyacidified, partially halogenated, or the like; cellulosic compounds,including, but not limited to, native or synthetic cellulose, cotton,regenerated cellulose (for example, rayon, cellophane, or the like),cellulose acetate, cellulose propionate, cellulose butyrate, celluloseacetate-propionate, cellulose acetate-butyrate, cellulosepropionate-butyrate, cellulose nitrate, methyl cellulose, ethylcellulose, carboxymethyl cellulose, carboxyethyl cellulose, cellulosesalts, and combinations or copolymers thereof, as naturally extracted,as synthesized, or as modified or processed in some way, includingpartially or fully esterified, partially or fully nitrated, partially orfully regenerated, partially or fully etherified, partially or fullyacidified, partially or fully acid-neutralized, or the like, orcombinations thereof; surface-active biomolecules or (co)polymers;poly(ethylene glycol) and/or poly(ethylene oxide) oligomers,homopolymers, or copolymers; autologous substances such as autologousbone marrow aspirates, autologous blood substances, or the like, or acombination thereof heterologous substances such as allogeneic bonemarrow aspirates, xenogenic bone marrow aspirates, allogeneic bloodsubstances, xenogenic blood substances, or the like, or a combinationthereof or the like, or combinations thereof. In an embodiment, the flowadditive comprises hyaluronic acid and/or a hyaluronate salt. In anotherembodiment, the flow additive comprises sodium hyaluronate. In analternate embodiment, the flow additive can include chondroitin,glucosamine, hyaluronic acid, a salt thereof, or a mixture thereof.

In one or more embodiments, an additive is included in the DBMcomposition to further modify the handling characteristics of thecomposition, such as viscosity and moldability. The additive may be abiocompatible polymer, such as a water-soluble cellulosic, or a naturalpolymer, such as gelatin. The additive may be added to either the dryDBM component or the liquid component. The additive may be used to atleast partially coat the DBM fibers prior to combining them with theliquid carrier. Non-limiting examples of additives suitable for use inthe DBM composition include gelatin, carboxymethyl cellulose,hydroxypropyl methylcellulose, methylcellulose, hydroxyethyl cellulose,other cellulose derivatives, alginate, hyaluronic acid, sodium salts,polyvinyl pyrrolidones, polyvinyl alcohol, arabic gum, guar gum, xanthamgum, chitosans, and poloxamers.

As previously indicated, the implantable composition of this disclosurecan be freshly prepared just by mixing desired quantities of thedemineralized fibrous bone elements, fluid carrier and optionalcomponent(s), if any, in any suitable sequence of separate mixing,adsorption, rehydration or drying operations or all at once. Thus, thedemineralized fibrous bone elements prepared by delipidation/terminalsterilization described herein can be mixed with the optionalingredients(s) and thereafter combined with the fluid carrier component,the demineralized fibrous bone elements can be mixed with the fluidcarrier followed by addition of the optional ingredient(s) or theoptional ingredients can be added to the fluid carrier followed byaddition of the demineralized fibrous bone elements. Variations of theseand other sequences of mixing are, of course, possible. In variousembodiments, the implantable composition can include non-fibrous boneelements. In other embodiments, the fibrous elements and fluid carrierare mixed substantially simultaneously such that the fibrous elements ofthe implantable composition are entangled and the non-fibrous boneelements are thoroughly mixed in the entangled fibrous bone elements.

The amount of demineralized bone fibers prepared bydelipidation/terminal sterilization described herein which can beincorporated into the implantable composition can vary widely withamounts of about 99% weight, about 95% by weight, about 90% by weight,about 85% by weight 70% by weight. In various embodiments, the amount ofthe non-fibrous bone elements which can be incorporated into theimplantable composition can vary widely with amounts from about 10 toabout 90 weight percent, and in some aspects, from about 20 to about 70weight percent. The ratio of fibrous to non-fibrous bone elements canvary between about 0.2:1 to about 1:0.2. The balance of the compositionbeing made up of fluid carrier and optional ingredient(s), if any.

The bone tissue composition may be completely insoluble or may be slowlysolubilized after implantation. Following implantation, the compositionmay resorb or degrade, remaining substantially intact for at least oneto seven days or for two or four weeks or longer and often longer than60 days. The composition may thus be resorbed prior to one week, twoweeks, three weeks, or other, permitting the entry of bone healingcells.

Covering Material for Contaminated Bone Tissue

In some embodiments, the covering utilized to contain the contaminatedbone tissue for further treatment may be used for retaining ofparticulate or morselized bone tissues (the substance provided in thecovering), optionally to provide a focus or concentration of biologicalactivity. In some embodiments, the covering may be used for maintainingmaterials (the substance provided in the covering) in spatial proximityto one another, possibly to provide a synergistic effect. In someembodiments, the delivery system may be used to control availability ofsubstances provided within the delivery system to cells and tissues of asurgical site over time. In some embodiments, the covering may be usedfor delivery through a limited opening, such as in minimally invasivesurgery or mini-open access. In some embodiments, the covering may beused to deliver morselized or particulated materials (the substanceprovided in the covering) in pre-measured amounts.

In various embodiments, the covering can contain a demineralizedallograft material. The covering limits, and in some embodimentseliminates bone tissue migration and maintains bone tissue density. Thecovering containing demineralized allograft material, may be configuredto conform to surrounding bony contours or implant space. In someembodiments, the delivery system provides a pathway for healing/cellpenetration and tissue ingrowth. Thus, the covering may facilitatetransfer of a substance out of the covering or transfer or surroundingmaterials at the surgical site, such as cells and tissues, into thecovering.

The covering useful to contain the contaminated bone tissue may have asingle compartment or may have a plurality of compartments. Thus, in oneembodiment, the covering is dual-compartment and comprises first andsecond compartments. A first substance may be provided in the firstcompartment and a second substance may be provided in the secondcompartment. The second compartment may be adjacent to, apart from,inside, or surrounding the first compartment. Materials forming thefirst compartment and the second compartment may be the same ordifferent. Selection of materials, positioning of the compartments, andother factors relating to the first and second compartments may bechosen to achieve simultaneous or sequential delivery or release of asubstance or substances.

The covering may comprise a structural material and, in someembodiments, a functional material. The structural material may comprisea mesh material, a polymeric material, or other. The functional materialmay comprise, for example, a radiopaque material, a bactericidalmaterial, or other material.

In various embodiments, in accordance with the specific application forwhich the covering is being used, the covering may be rigid, may beflexible, may be non-elastic, or may be elastic. The covering materialmay be braided, woven, non-woven shape memory, particulate, threaded,porous, or non-porous.

The covering may participate in, control, or otherwise adjust therelease of the substance. For example, the covering may act as aselectively permeable membrane and/or may be porous, with the level ofporosity being related to the nature of the substances inside thecovering. Thus, the material for and configuration of the covering maybe selected or adjusted based on desired release characteristics.Specific properties that may be adjusted include thickness,permeability, porosity, strength, flexibility, elasticity, and others ofthe covering material. It is to be appreciated that some of theseproperties may depend on others. For example, the thickness and porosityof the material may contribute to its strength, flexibility, andelasticity.

In some embodiments, the covering may be porous to fluid and/or cells,may be biocompatible, and may be resistant to rupture (including shouldthe substance provided therein swell). In some embodiments, the coveringwith the demineralized allograft material provided therein may beloadbearing. The covering may be resorbable or non-resorbable. Thecovering may provide increased handling properties, may have irrigationresistance, and/or may support cellular penetration. Flexibility of thecovering may be selected to suit particular applications. In someapplications, it may be desirable to have a flexible covering.

If the covering is made from a resorbable material, the coveringdegrades and disappears after a period of time. If the covering is notmade of a resorbable material, the covering remains in the body. Tissueingrowth may occur to bind the host tissue to the substance providedwithin the covering. Tissue ingrowth through and around the covering,between the host tissue and the substance provided within the covering,may be promoted via openings in the covering.

In various embodiments, the covering may comprise a porous material or amesh material. The size of the pores of the covering may be designed topermit cellular infiltration (approximately several microns to severalmillimeters), but may also be designed specifically to exclude cells forthe inside of the covering (e.g. approximately 0.45 microns) and onlyallow diffusion of small molecules (proteins and hormones). Thus, thecovering may act to control access to the interior of the deliverysystem by cells. In embodiments comprising more than one compartment,characteristics of the covering material may be varied betweencompartments. Generally, the porosity, flexibility, strength, or anyother characteristic of one compartment may vary from thatcharacteristic of the other compartment.

The covering may be formed of a resorbable or non-resorbable, natural orsynthetic biocompatible material. In some embodiments, more than onematerial may be used, including as multiple layers. For example, in anembodiment comprising two compartments, one or more materials may beused for the first compartment and a different material or materials maybe used for the second compartment. For example, one compartment orportions thereof may be made of material or materials that provide adesired property or properties relative to other compartments orportions thereof, such as increased or decreased resorbability orstiffness, or the different compartments or portions thereof may beimparted with different drug delivery properties, etc. Alternatively,all compartments may comprise the same material or mixtures ofmaterials. Where the characteristics of the material are varied betweencompartments or over the surface of a single compartment, the pores ofthe first compartment or portion thereof may be larger than the pores ofthe second compartment.

The covering may comprise any suitable structure for delivering asubstance in vivo. Thus, as described, the covering may comprise a mesh.In other embodiments, the covering may comprise a polymeric structurewith a chamber provided therein. The chamber may be filled with asubstance for delivering in vivo, such as demineralized allograftmaterial, fully mineralized bone tissue, or others disclosed herein.

In some embodiments, the covering may expand when placed in the body.Expansion can be provided in at least two ways: the covering may becompressed such that the covering expands when placed in the body or thecovering may be made of a material that expands when it comes in contactwith water or other bodily fluids, either by way of liquid absorption orby stretching when the materials inside it absorb liquid and themselvesexpand. In some embodiments, the covering may comprise a shape memorymaterial such as copper-zinc-aluminum-nickel alloy,copper-aluminum-nickel alloy, and nickel-titanium (NiTi) alloy.Reinforcing materials such as cortical bone, calcium phosphates, etc.may be incorporated into the structure of the covering to reinforce it.

The covering may be configured for specific compressive strength andrigidity by adjusting density and resorption time of the covering. Insome embodiments, a coating may be provided over the covering. Forexample, the coating may be a compound of poly-L-lactide, ofpolyglycolic acid, or their polymers. The coating may be selected suchthat it has a resorption time wherein it is resorbed by the body and thematerial within the covering is permitted to exit through openings inthe covering.

Exemplary Covering Materials

A covering according to an aspect of the present disclosure may comprisedemineralized allograft material and at least one of bioerodiblepolymers, bioabsorbable polymers, biodegradable biopolymers, syntheticpolymers, copolymers and copolymer blends and combinations thereof.Exemplary materials may include biopolymers and synthetic polymers suchas human skin, human hair, bone sheets, collagen, fat, thin cross-linkedsheets containing fibers and/or fibers and chips, degradable sheets madefrom polyethylene glycol (PEG), chitosan sheets, alginate sheets,cellulose sheets, hyaluronic acid sheet, as well as copolymer blends ofpoly (lactide-co-glycolide) PLGA.

Exemplary materials may include polymeric material, woven material andbraided material, non-woven; shape memory material; using outerparticles to contain inner particles; attach particles to threads; addporosity to mesh fibers; non-porous materials; non-porous materials. Insome embodiments, materials may be used for portions of the covering,such as for a compartment of the covering that is substantiallyimpenetrable.

In some embodiments, the covering may comprise a mesh material. Suitablemesh materials include natural materials, synthetic polymeric resorbablematerials, synthetic polymeric non-resorbable materials, and othermaterials. Natural mesh materials include silk, extracellular matrix(such as DBM, collagen, ligament, tendon tissue, or other),silk-elastin, elastin, collagen, and cellulose. Synthetic polymericresorbable materials include poly (lactic acid) (PLA), poly (glycolicacid) (PGA), poly (lactic acid-glycolic acid) (PLGA), polydioxanone,PVA, polyurethanes, polycarbonates, and others. Other suitable materialsinclude carbon fiber, metal fiber, and various meshes. In otherembodiments, the covering may comprise non-woven material such as a spuncocoon or shape memory materials having a coil shape or shape memoryalloys.

Generally, the covering may be formed of any natural or syntheticstructure (tissue, protein, carbohydrate) that can be used to form acovering configuration. Thus, the covering may be formed of a polymer(such as polyalkylenes (e.g., polyethylenes, polypropylenes, etc.),polyamides, polyesters, poly(glaxanone), poly(orthoesters),poly(pyrolicacid), poly(phosphazenes), polycarbonate, otherbioabsorbable polymer such as Dacron or other known surgical plastics, anatural biologically derived material such as collagen, gelatin,chitosan, alginate, a ceramic (with bone-growth enhancers,hydroxyapatite, etc.), PEEK (polyether-etherketone), dessicatedbiodegradable material, metal, composite materials, a biocompatibletextile (e.g., cotton, silk, linen), extracellular matrix components,tissues, or composites of synthetic and natural materials, or other.Various collagen materials can be used, alone or in combination withother materials, including collagen sutures and threads. Any suitablecollagen material may be used, including known collagen materials. Someexamples include polymer or collagen threads woven, or knitted into amesh. Other suitable materials include thin polymer sheets molded in thepresence of a porogen and having underwent leaching; polymer sheets ornaturally derived sheets such as fascia and other collagen materials,small intestinal submucosa, or urinary bladder epithelium, the sheetsbeing punctured to introduce porosity; specific shapes printed usingavailable or future printing technologies; naturally secreted materialssuch as bacterial cellulose grown within specific molds; etc.

In some embodiments, mesh fibers may be treated to impart porosity tothe demineralized allograft material that is in fiber form. This may bedone, for example, to PLA, PLGA, PGA, and other fibers. One suitablemethod for treating the mesh fibers comprises supercritical carbondioxide, supercritical nitrogen, or supercritical water treatment topartially solubilize the particles. This treatment may further becarried out for viral inactivation. Another suitable method for treatingthe mesh fibers comprises explosive decompression. Explosivedecompression generates porosity and leads to controlled permeability.The mesh material further may be loaded with cells, growth factors, orbioactive agents.

In further embodiments, fibers of a mesh material may be treated such asby having particles adhered thereto. The particles may be, for example,bone particles, demineralized allograft material, or the like. Thus, inone embodiment, the covering may comprise a plurality of threads formedinto a fabric. The threads may have particles adhered thereto. Forexample, the threads may have particles strung on the thread. In analternative embodiment, the covering may be formed of a material and thematerial may be coated with particles.

In yet other embodiments, the covering may comprise a non-porousmaterial, which may be permeable. A non-porous material may be used forlater (or delayed) delivery of a substance provided therein. Suchsubstance may comprise, for example, cells, growth factors, or bonemorphogenetic proteins. Accordingly, in one embodiment, a deliverysystem for delayed delivery of cells, growth factors, or bonemorphogenetic proteins is provided comprising a non-porous covering.

In particular, in various embodiments, the device may comprise abioerodible, a bioabsorbable, and/or a biodegradable biopolymer that mayprovide immediate release, or sustained release of the clonidine.Examples of suitable sustained release biopolymers include but are notlimited to poly (alpha-hydroxy acids), poly (lactide-co-glycolide)(PLGA), polylactide (PLA), polyglycolide (PG), polyethylene glycol (PEG)conjugates of poly (alpha-hydroxy acids), poly(orthoester)s (POE),polyaspirins, polyphosphagenes, collagen, starch, pre-gelatinizedstarch, hyaluronic acid, chitosans, gelatin, alginates, albumin, fibrin,vitamin E compounds, such as alpha tocopheryl acetate, d-alphatocopheryl succinate, D,L-lactide, or L-lactide, -caprolactone,dextrans, vinylpyrrolidone, polyvinyl alcohol (PVA), PVA-g-PLGA,PEGT-PBT copolymer (polyactive), PEO-PPO-PAA copolymers, PLGA-PEO-PLGA,PEG-PLG, PLA-PLGA, poloxamer 407, PEG-PLGA-PEG triblock copolymers, SAIB(sucrose acetate isobutyrate) or combinations thereof. As persons ofordinary skill are aware, mPEG and/or PEG may be used as a plasticizerfor PLGA, but other polymers/excipients may be used to achieve the sameeffect. mPEG imparts malleability to the resulting formulations. In someembodiments, these biopolymers may also be coated on a medical device toprovide the desired release profile. In some embodiments, the coatingthickness may be thin, for example, from about 5, 10, 15, 20, 25, 30,35, 40, 45 or 50 microns to thicker coatings 60, 65, 70, 75, 80, 85, 90,95, 100 microns to delay release of the substance from the medicaldevice. In some embodiments, the range of the coating on the medicaldevice ranges from about 5 microns to about 250 microns or 5 microns toabout 200 microns to delay release from the medical device.

Functional Material Characteristics

The covering material may have functional characteristics.Alternatively, other materials having functional characteristics may beincorporated into the covering in addition to bone tissue. Functionalcharacteristics may include radiopacity, bacteriocidity, source forreleased materials, tackiness, or the like. Such characteristics may beimparted substantially throughout the covering or at only certainpositions or portions of the covering.

Suitable radiopaque materials include, for example, ceramics,mineralized bone, ceramics/calcium phosphates/calcium sulfates, metalparticles, fibers, and iodinated polymer. Polymeric materials may beused to form the covering and be made radiopaque by iodinating them.Other techniques for incorporating a biocompatible metal or metal saltinto a polymer to increase radiopacity of the polymer may also be used.Suitable bacteriocidal materials may include, for example, tracemetallic elements. In some embodiments, trace metallic elements may alsoencourage bone growth.

Functional material, such as radiopaque markers, may be provided at oneor more locations on the covering or may be provided substantiallythroughout the covering. Thus, for example, in a tubular covering, aradiopaque marker may be provided at a tip of the tubular covering. Suchmarker may facilitate placement of the covering. Radiopaque materialsmay be incorporated into the covering and/or into the substance fordelivery by the covering. Further, radiopaque materials may be providedat only some locations on the covering such that visualization of thoselocations provides indication of the orientation of the covering invivo.

The covering itself may be designed to release materials duringdegradation of the covering material. Thus, bone morphogenetic proteins(BMPs), growth factors, antibiotics, angiogenesis promoting materials(discussed more fully below), bioactive agents (discussed more fullybelow), or other actively releasing materials may be incorporated intothe covering material such that as the covering material is degraded inthe body, the actively releasing material is released. For example, anactively releasing material may be incorporated into a biodegradablepolymer covering such as one manufactured of a biodegradable polyestersuch as poly(lactic acid) (PLA), poly(glycolic acid) (PGA),poly(lactic-co-glycolic acid) (PLGA), or polyhydroxyalkanoates(polyhydroxybutyrates and polyhydroxyvalerates and copolymers). In someembodiments, poly(ethylene glycol) (PEG) may be incorporated into thebiodegradable polyester to add hydrophilic and other physico-chemicalproperties to enhance drug delivery. In some embodiments, composites ofallograft bone and biodegradable polymers (for example, PLEXOR® productsavailable from Osteotech™) may be used in the covering.

In some embodiments, the covering may comprise a material that becomestacky upon wetting. Such material may be, for example, a protein orgelatin based material. Tissue adhesives, including mussel adhesiveproteins and cyanoacrylates, may be used to impart tackiness to thecovering. In further examples, alginate or chitosan material may be usedto impart tackiness to the covering. In further embodiments, an adhesivesubstance or material may be placed on a portion of the covering or in aparticular region of the covering to anchor that portion or region ofthe covering in place at an implant site.

It will be understood that various modifications may be made to theembodiments disclosed herein. Therefore, the above description shouldnot be construed as limiting, but merely as exemplification of thevarious embodiments. Those skilled in the art will envision othermodifications within the scope and spirit of the claims appended hereto.

What is claimed is:
 1. A method of decontaminating bone tissue, themethod comprising: contacting the bone tissue having contaminantstherein with carbon dioxide to decontaminate the bone tissue and formcarbon dioxide having contaminants; collecting the carbon dioxide havingcontaminants; and separating the contaminants from the collected carbondioxide.
 2. A method of decontaminating bone tissue of claim 1,comprising treating the contaminated bone tissue with ethanol gradientdehydration.
 3. A method of decontaminating bone tissue of claim 2,further comprising flushing with liquid carbon dioxide the contaminatedbone tissue treated with ethanol gradient dehydration.
 4. A method ofdecontaminating bone tissue of claim 3, further comprising subjectingthe contaminated bone tissue to critical point dehydration and/orsupercritical carbon dioxide treatment; and releasing the carbon dioxideat a controlled rate.
 5. A method of decontaminating bone tissue ofclaim 4, wherein the temperature and pressure of carbon dioxide israised to or above 31.1° C. and 1100 psi.
 6. A method of decontaminatingbone tissue of claim 4, wherein supercritical carbon dioxide treatmentis carried out at approximately 105° C. and approximately 7000 psi.
 7. Amethod of decontaminating bone tissue of claim 1, further comprisingremoving the contaminated carbon dioxide, purifying the contaminatedcarbon dioxide with supercritical fluid, liquefying the purified carbondioxide and using liquid purified carbon dioxide as a cooling fluid. 8.A method of decontaminating bone tissue of claim 1, wherein contaminatedcarbon dioxide is separated by bubbling through water and/or an organicsolvent to remove contaminants comprising lipids, disease causingpathogens, viruses or bacteria.
 9. A method of decontaminating bonetissue of claim 8, further comprising treating the carbon dioxidebubbled through water and/or the organic solvent with acid treatment,filtering and liquefying the carbon dioxide.
 10. A method ofdecontaminating bone tissue of claim 9, wherein the liquefied carbondioxide is purified to 99.9% free of lipids, disease causing pathogens,viruses or bacteria.
 11. A method of decontaminating bone tissue ofclaim 9, further comprising generating processing charts includingvanishing interfacial tension and carbon dioxide pressure.
 12. A methodof decontaminating bone tissue of claim 1, wherein the bone tissuecomprises bone fibers, bone chips, bone particles, bone matrices ormixtures thereof.
 13. A method of decontaminating bone tissue of claim1, further comprising providing a delivery vehicle for the bone tissuehaving contaminants, the delivery vehicle comprising a carrier orcovering.
 14. A method of decontaminating bone tissue of claim 1,wherein the method of decontaminating the bone tissue is a multiplebatch process.
 15. A method for purifying contaminated carbon dioxidefrom contaminated bone tissue, the method comprising: collectingcontaminated carbon dioxide from carbon dioxide treated contaminatedbone tissue; separating contaminants from the collected contaminatedcarbon dioxide to obtain purified carbon dioxide.
 16. A method forpurifying carbon dioxide from contaminated bone tissue of claim 15,wherein the contaminated carbon dioxide is separated from thecontaminants by bubbling the contaminated carbon dioxide through waterand/or an organic solvent to remove the contaminants comprising lipids,disease causing pathogens, viruses or bacteria.
 17. A method forpurifying carbon dioxide from contaminated bone tissue of claim 16,further comprising treating the carbon dioxide bubbled through waterand/or the organic solvent with acid treatment, filtering and liquefyingthe carbon dioxide.
 18. A method for purifying carbon dioxide fromcontaminated bone tissue of claim 17, wherein the liquefied carbondioxide is purified to 99.9% free of lipids, disease causing pathogens,viruses or bacteria.
 19. A system for treating contaminated bone tissuewith purified carbon dioxide, the system comprising a bone tissuechamber configured for holding contaminated bone tissue and receivingpurified carbon dioxide into the bone tissue chamber and evacuatingcontaminated carbon dioxide from the bone tissue chamber; a purifiedcarbon dioxide supply configured for supplying purified carbon dioxideto the bone tissue chamber to decontaminate the bone tissue; acollection chamber configured to receive contaminated carbon dioxidefrom the bone tissue chamber and evacuate contaminated carbon dioxidefrom the collection chamber; and a purification chamber configured toreceive contaminated carbon dioxide from the collection chamber and toremove contaminants from the contaminated carbon dioxide by apurification material to obtain purified carbon dioxide, thepurification chamber configured to evacuate the purified carbon dioxideand supply it to the bone tissue chamber.
 20. A system for treatingcontaminated bone tissue with purified carbon dioxide of claim 19,further comprising a controller and a signal transmission systemfunctionally interconnecting the controller with the bone tissuechamber, the purified carbon dioxide supply, the collection chamber andthe purification chamber, the controller comprising computer readableinstructions to cause the controller to effect the evacuation ofcontaminated carbon dioxide from the bone tissue chamber to thecollection chamber, send the contaminated carbon dioxide to thepurification chamber, and to dispense the purified carbon dioxide to thebone tissue chamber.